We present patient characteristics and follow-up results of cases with anterior chamber dexamethasone implant migration. The common feature of all six presented cases was vitrectomized eyes. Four of the patients had sutured intraocular lens (IOL) implantation due to complicated cataract surgery, one had combined retinal detachment surgery with sutured IOL implantation, and one had vitrectomy for diabetic intravitreal hemorrhage cleaning and uncomplicated cataract surgery. Anterior chamber implant migration caused corneal edema in all cases and elevated intraocular pressure in three cases. In two cases, the dexamethasone implant was directed into the vitreous cavity after maximum pupillary dilation and corneal manipulation with cotton tip applicator with the patient in reverse Trendelenburg position. There was no other complication until dexamethasone implant degradation, with clear cornea at final examination. In two cases, the implant was removed from the anterior chamber by aspiration, but keratoplasty surgery was planned due to endothelial cell loss and persistent corneal edema during follow-up. In the last two cases, the dexamethasone implant was redirected into the vitreous chamber with a 23-gauge catheter and anterior chamber maintainer but they migrated into the anterior chamber again. In one of these patients, the implant was aspirated by catheter and corneal transplantation was performed due to corneal edema, while the other patient’s implant was redirected into the vitreous chamber with no further anterior migration. The risk of dexamethasone implants migrating into the anterior chamber of vitrectomized eyes and those with sutured IOL implantation should be kept in mind and the patient should be informed and advised to see an ophthalmologist immediately before permanent corneal endothelial damage occurs. The most common complication reported after dexamethasone implantation is an increase in intraocular pressure, which peaks at about 3 months.5 In addition to the side effects of dexamethasone reported in the literature, such as cataracts and increased intraocular pressure, the implantation procedure itself involves the risk of complications like dislocation to the anterior chamber, corneal endothelial damage, secondary corneal edema, and implantation in the lens.8 Migration of a dexamethasone implant into the anterior chamber is a rare complication that can be managed by directing the implant back into the vitreous cavity or removing it from the anterior chamber through a corneal incision.10 In this series of six cases, we discuss risk factors, clinical course, and treatment approaches for migration of dexamethasone implants to the anterior chamber.Ozurdex  is a rod-shaped, biodegradable dexamethasone implant 6 mm in length and 0.46 mm in diameter that is injected into the intravitreal cavity using a 22-gauge needle. It is effective in the treatment of macular edema due to retinal vein occlusion, non-infectious uveitis affecting the posterior segment, and diabetic macular edema.Patients who underwent dexamethasone implantation in the ophthalmology department of Manisa Celal Bayar University Faculty of Medicine within the past 5 years and had anterior chamber dislocation of the implants during follow-up are discussed in terms of etiologies, treatment approaches, and outcomes.A 63-year-old man was being followed in our retina unit after undergoing pars plana vitrectomy (PPV) with silicone oil injection due to retinal detachment in the left eye, followed by silicone removal 4 months later. At last examination, his Snellen visual acuity was 0.7 in the right eye and 0.15 in the left eye. Intraocular pressure (IOP) was 14 mmHg in the right eye and 16 mmHg in the left eye. On slit-lamp examination, nuclear cataract was observed in the right eye, while pseudophakia, posterior capsule defect, and zonular dialysis were observed in the left eye. Fundus examination was normal in the right eye and showed attached retina and peripheral cryotherapy and laser scars in the left eye. On optical coherence tomography (OCT) imaging, the right eye appeared normal, while cystoid macular edema  was observed in the left eye . Fundus A 60-year-old woman who was referred to our clinic from another center due to complicated cataract surgery underwent PPV and sutured intraocular lens (IOL) implantation. In the last examination, her Snellen visual acuity was 0.1 in the right eye and 1.0 in the left eye, while IOP was 19 mmHg in the right eye and 18 mmHg in the left eye. On fundus examination, the macula was elevated and optic disc was normal in the right eye, while the left eye was entirely normal. OCT revealed cystoid edema in the right eye and normal findings in the left eye. FFA imaging in the right eye showed late-phase hyperfluorescence in the macula, and it was decided to treat her right eye with dexamethasone implant. In examination performed 2 weeks after implantation, the right eye exhibited mild corneal edema and the dexamethasone implant was visible in the anterior chamber . Fundus A 61-year-old man with proliferative diabetic retinopathy who underwent PPV, endolaser, and phacoemulsification with IOL implantation in the right eye and PPV and endolaser in the left eye due to bilateral intravitreal hemorrhage was being followed up in our retina unit. At last examination, visual acuity was 0.05 cm in the right eye and counting fingers at 50 cm in the left eye, while intraocular pressure was 17 mmHg in the right eye and 16 mmHg in the left eye. Slit-lamp examination revealed pseudophakia and clear cornea in the right eye and posterior subcapsular cataract in the left eye. On fundus examination, cellophane maculopathy, laser scars in the periphery, and macular edema were observed in the right eye. Laser scars and macular microhemorrhages were observed in the left eye. Bilateral cystoid macular edema was noted on OCT. No response was achieved despite 4 bilateral intravitreal ranibizumab injections, so the decision was made to administer dexamethasone implants. Slit-lamp examination at 1-month follow-up after dexamethasone implantation revealed corneal edema and the dexamethasone implant was seen in the anterior chamber of the right eye . Using aA 79-year-old woman who was referred to our clinic from another center for complicated cataract surgery underwent left PPV and sutured IOL implantation. At last examination, her Snellen visual acuity was 0.6 in the right eye and counting fingers at 3 m in the left eye; IOP was 16 mmHg in the right eye and 14 mmHg in the left eye. Both eyes were found to be pseudophakic on slit-lamp examination. Fundus examination revealed irregularity of the retinal pigment epithelium in the right eye and laser scars in the inferior hemisphere of the left eye. OCT findings were normal in the right eye, while spongy edema was observed in the left eye. FFA imaging showed macular edema with traction in the inferotemporal quadrant of the left eye, and it was decided to administer a dexamethasone implantation. The implant appeared in the anterior chamber 15 days later . After pA 73-year-old man who was referred to our clinic from another center due to traumatic cataract and zonular dialysis underwent left PPV and anterior vitrectomy, followed by scleral-fixated IOL implantation 2 months later. In ophthalmologic examination at postoperative 2 months, his Snellen visual acuity was 0.9 in the right eye and counting fingers at 1 m in the left eye, while IOP was 12 mmHg in the right eye and 18 mmHg in the left eye. Both eyes appeared pseudophakic on slit-lamp examination. Fundus examination revealed macular retinal pigment epithelial irregularity in the macula in both eyes. On OCT, the right eye was normal, while macular edema (447 µm) was observed in the left eye. The right eye showed no leakage on FFA imaging, while the left eye showed macular edema and areas of hyperfluorescence. A dexamethasone implant was injected into the left eye. The patient presented to the clinic 25 days after implantation with further visual deterioration (hand movements). The implant was observed in the anterior chamber of the left eye on slit-lamp examination . Using aA 70-year-old man who was being followed up in our clinic for angle-closure glaucoma underwent left PPV and phacoemulsification, followed by scleral-fixated IOL implantation 6 months later. At last examination, his Snellen visual acuity was 1.0 in the right eye and 0.2 in the left eye; IOP was 12 mmHg in the right eye and 17 mmHg in the left eye. On slit-lamp examination, shallow anterior chamber and patent iridotomy were observed in the right eye and clear cornea, iridotomy, centered IOL, and iris atrophy in the superior quadrant were observed in the left eye. On fundus examination, cup-to-disc ratio was normal in the right eye and 0.3 in the left eye. OCT findings were normal in the right eye, while macular edema (434 µm) was present in the left eye. On FFA imaging, there was no leakage in the right eye, while the left eye showed macular edema with late-phase hyperfluorescent areas. The patient received 5 intravitreal ranibizumab injections. Due to macular edema (550 µm), it was decided to administer a dexamethasone implant. Twenty days after implantation, the patient presented to the clinic with complaints of vision loss. His visual acuity was at the level of hand movements in the left eye, and slit-lamp examination revealed corneal edema and the dexamethasone implant in the anterior chamber . His IOPThe characteristics and outcomes of patients with dexamethasone implant migration from the vitreous cavity to the anterior chamber are presented in 11 Long plane journeys within the first week after dexamethasone implantation were also reported to be a risk factor for anterior chamber dislocation by increasing vitreous pressure due to changes in air pressure.12 The clinical findings and risk factors of our patients were consistent with the information in the literature. All of our patients had undergone PPV and had posterior capsule defect and/or zonular dialysis.In this study, we present information pertaining to patients who were followed up and treated for dislocation of dexamethasone implants into the anterior chamber. The patients included 3 women and 3 men between the ages of 60 and 79 years. All of the affected eyes were vitrectomized and pseudophakic. One patient (Case 1) had posterior capsule defect, zonular dialysis, and IOL implantation in the sulcus, 4 patients  had undergone sutured IOL implantation after complicated cataract surgery, and 1 patient (Case 3) had undergone intracapsular IOL implantation with no posterior capsule defect. Anterior chamber migration of a dexamethasone implant is a rare complication and risk factors include previous vitrectomy, aphakic eyes, posterior capsule opening, and lying in prone position.13 In addition, spontaneous return of the implant to the vitreous cavity has also been reported.14 The procedure of guiding the implant back into the vitreous cavity by placing the patient in supine position after pupil dilation was first described by Kishore and Schaal.9 Mateo et al.15 reported scleral fixation of the dexamethasone implant using a 10-0 suture. In 2 of our patients with dexamethasone implant in the anterior chamber (patients 2 and 4), the patients were placed in reverse Trendelenburg position after pharmacological dilation and the implant was guided back into the vitreous cavity by manipulating the cornea with a sterile cotton tip applicator. In 2 other patients (patient 3 and patient 5 after first migration), the implant was moved from the anterior chamber back into the vitreous cavity using a 23-G catheter and anterior chamber maintainer. In the patients with severe corneal edema and elevated IOP , the dexamethasone implant was removed from the anterior chamber using a 23-G catheter. One of the 2 dexamethasone implants detected in the anterior chamber in patient 1 was implanted in our center, while the other was most likely implanted as a second dose within a short period at another center. This could not be explained conclusively.In the event of a dexamethasone implant in the anterior chamber, the implant can be removed from the anterior chamber via a corneal incision or directed back into the vitreous cavity.7 reported that corneal edema developed when anterior chamber migration occurred within the first 3 weeks after dexamethasone implantation, but did develop in migrations occurring between 5 weeks and 3 months after implantation. All of the patients in our series presented with early migration, within 1 month of implantation, and severe corneal edema was observed in 3 of the patients . Keratoplasty was planned for patient 6 and patient 1, who had 2 implants in the anterior chamber, due to persistent bullous corneal changes and very low endothelial cell counts. Patient 5 underwent keratoplasty due to bullous keratopathy and corneal endothelial failure. The patients who did not develop corneal edema were those who presented to our clinic promptly after onset of their complaints and received rapid intervention.Khurana et al.16 retrospectively analyzed 924 cases of intravitreal dexamethasone injection. Anterior chamber migration of the implant occurred in 4 patients within 2 to 6 weeks. In 2 patients, the implant was guided back into the vitreous cavity. In the other 2 patients, implant migration occured twice in one and 3 times in the other before the implants were surgically removed. One of the patients underwent keratoplasty. All of the affected eyes lacked posterior capsule integrity.16 In our case series, 2 dexamethasone implants were detected in the anterior chamber of 1 patient (Case 1). They were removed from the anterior chamber by aspiration using a 23-gauge catheter. Keratoplasty was planned for this patient in the cornea unit because he developed bullous keratopathy and corneal endothelial decompensation. In the case of 2 other patients (Cases 2 and 4), the implant was maneuvered back into the vitreous cavity by applying pressure to the cornea with a sterile cotton tip applicator with the patient in supine position after pupil dilation and reverse Trendelenburg positioning. This resulted in no further problems until implant degradation and the cornea was clear at final examination. In 2 of our patients (Cases 3 and 5), the implant was removed from the anterior chamber and surgically repositioned in the vitreous cavity using a 23-G catheter and anterior chamber maintainer. However, in both patients, repeat migration of the dexamethasone implant was observed. In 1 of these patients (Case 3), the implant was surgically repositioned again with no further problems, while in the other patient (Case 5) explantation was performed using a 23-G catheter. This patient later underwent keratoplasty due to corneal endothelial failure and bullous keratopathy. The implant was also explanted from another patient (Case 6) using a 23-G catheter. Keratoplasty was planned in the cornea unit due to the development of corneal endothelial failure and bullous keratopathy. In all of our patients, the dexamethasone implant migrated into the anterior chamber within 1 to 4 weeks of implantation. Five patients  lacked posterior capsule integrity and all patients had undergone PPV.Kang et al.17 retrospectively analyzed 468 patients who received dexamethasone implant injections at multiple centers and determined the prevalence of implant migration to be 1.6%. They also reported a significant relationship between implant migration and cataract surgery (p=0.043), intraocular lens status (p=0.005), and vitrectomy (p=0.057). Öner et al.18 injected a dexamethasone implant for macular edema in a patient who underwent PPV and scleral-fixated IOL implantation after complicated cataract surgery. Fifteen days after the implantation, the patient exhibited corneal edema and anterior chamber migration of the dexamethasone implant. Explantation was performed, but corneal edema persisted at 4-month follow-up. All of our patients had history of cataract surgery and vitrectomy. Four had sutured intraocular lenses, 1 had an open posterior capsule, zonular dialysis, and a sulcus lens, and 3 patients developed bullous keratopathy.Goncalves et al.In cases of anterior chamber dislocation of dexamethasone implants, the implant should be removed or repositioned in the vitreous cavity as soon as possible in order to prevent permanent corneal edema due to corneal endothelial damage. Pupil dilation and reverse Trendelenburg positioning followed by positional guidance of the implant toward the vitreous by cornea manipulation with a sterile cotton tip applicator is a noninvasive procedure that can be used as a first approach in suitable patients. Patients should be advised to avoid long trips and the prone position after dexamethasone implantation and to see an ophthalmologist immediately if they experience any ocular complaints.Risk factors for anterior chamber migration of dexamethasone implant such as PPV, previous complicated cataract surgery, lack of posterior capsule integrity, and zonular dialysis should be evaluated carefully and implantation should be avoided in patients who are at risk.


In July 2019, a PRRSV-negative boar station was infected with a recombinant of two PRRSV vaccine strains, which subsequently spread to at least 36 herds that had received semen from the boar station. In the following months, all the infected herds reported reduced productivity. The aim of the present study was to evaluate the impact of the PRRS outbreak.Production data were collected from 13 of the herds. The average levels of farrowings/week, liveborns/litter, stillborns/litter, pre-weaning mortality and weaned pigs/litter were compared for the five-month period after infection and the preceding 7 months before infection with the new variant of PRRSV-1. Twelve herds experienced a decrease in farrowings/week (0.1–10.8% fewer farrowings/week), and all herds experienced fewer liveborns (0.8–4.8 fewer liveborns/litter) and more stillborns (0.6–2.6 more stillborns/litter). Pre-weaning mortality nearly doubled in half of the herds. Overall, the 13 herds were missing 2.4–6.5 pigs/litter at weaning during the 5 months after infection compared to the seven preceding months before infection.In this study, the impact of this new PRRSV-1 variant on productivity exceeded that typically seen in Danish herds infected with PRRSV-1. Since it first appeared at the beginning of the 1990s, porcine reproductive and respiratory syndrome virus (PRRSV) has been one of the major health challenges in pig production. According to figures from the Danish SPF society, approximately 35% of Danish pig herds are positive for antibodies to PRRSV, although the prevalence of serologically positive herds is declining [In 1994, production losses due to acute infections with PRRS in 30 Danish herds were estimated to be 1.2 pigs/year [Limited surveillance of the genetic diversity is practised in Denmark, but the available data indicate that, until the summer of 2019, two major clades of PRRSV-1 were co-circulating . One of In July 2019, PRRSV-1 was detected in samples taken as part of the routine PRRSV surveillance in one of the Danish PRRSV-negative boar stations. More than 70 breeding and multiplier herds and up to 700 production herds were at risk of having received semen from the most likely time of infection of the boar station and until the boar station was closed. Indeed, the virus was shortly after detected in three breeding herds and in at least 33 production herds that had received semen from this station. Subjective preliminary reports from the veterinarians consulting the herds and the herd owners indicated that the virus induced clinical signs similar to, or even exceeding, those normally observed in Danish herds infected with PRRSV-1. The clinical signs included sustained reproductive failures and high piglet mortality.The aim of the present study was to evaluate the impact of the outbreak in the infected herds over a period of 5 months after infection compared to the preceding 7 months. Furthermore, the production losses were compared to previous studies on the impact of PRRSV in Danish herds.Only 18 herd owners responded to the invitation letter, three of whom did not want to share data and two had given up recording data on productivity due to the psychological pressure caused by all the dead piglets. Therefore, we managed to include data from a total of 13 herds infected with the new PRRSV-1 variant in July 2019.The herd size ranged from 324 to 1650 sows (1009 median). The declared health status reported by SPF-SuS [Records from VETSTAT (the Danish system for surveillance of the veterinary use of drugs) showed that, prior to the infection with the recombinant PRRSV-strain, only one herd had mass-vaccinated the sows with “Porcilis® PRRS VET” . After infection with the new PRRSV-1 variant, most of the herd owners chose to mass-vaccinate with a modified live PRRS vaccine (MLV) in an attempt to control the new PRRSV-1 variant. Most of the herds used the MLV “Unistrain PRRS” , three herds used the MLV “Porcilis® PRRS VET”, and one herd used the killed-virus vaccine “Progressis® Vet”   and the Suvaxzyn PRRSV  in that the ORF 1, ORF 2 and part of ORF 3 are more than 99% identical to the Suvaxyn strain, and the rest of the genome is more than 99% identical to the Unistrain vaccine strain. The breakpoint is located in ORF 3 encoding the glycoprotein 3 (GP3) after nucleotide position 201, corresponding to amino acid 67 . It is nFor ten of the herds, the health status reported by SPF-SuS  was negaRecords from VETSTAT  show thaThe number of farrowings per week was affected by infection with the new PRRSV-1 variant, with 12 herds experiencing a reduction, which is expected after infection with PRRSV. One herd had a small increase in farrowings per week, which was probably due to the fact that relatively more sows were inseminated after infection in order to compensate for a potential decrease in farrowings as a result of PRRSV.Since PRRSV affects both the survival of the piglets and the reproduction of the sow, it is still too early to analyse the overall effects of PRRSV-1 infection on the reproductive parameters (e.g. wastage days and farrowing percentage), and therefore only effects including farrowings/week, liveborns/week, stillborns/week, pre-weaning mortality and weaned pigs/litter were included in the analysis.The closest study to the one reported here seems to be the Danish study from 2013, which included eight herds. Seven herds were previously PRRS-free, and, of these, three were infected with PRRSV-1 and four with PRRSV-2. One herd had previously been infected with PRRSV-1 and had experienced an acute outbreak after introduction of PRRSV-2. The period with reduced production was estimated to be between ten and 90 weeks [All 13 herds experienced fewer liveborn pigs at farrowing, which was also expected after an outbreak of PRRS. The average decrease in liveborn pigs per litter was 2.8 pigs for every litter during the five-month period after infection compared to the preceding 7 months. When comparing this with the previous Danish estimate from 2013 [In addition to there being fewer liveborn pigs, there was also an average increase in stillborn pigs per litter of 1.4 for the 5 months after infection compared to the preceding 7 months. In the 2013 study, there was an average increase in stillborns per litter of just 0.3 during the seven-month period after infection with PRRSV. Thus, infection with the new PRRS1-variant appears to result in one more stillborn pig per litter compared to the previous outbreaks with other variants.The pre-weaning mortality increased to an average of 40% after infection with the new PRRSV-1 variant. The increased pre-weaning mortality was also reflected in a lower number of weaned pigs from each litter in all 13 herds. On average, the 13 herds were missing 4.1 pigs from each litter at weaning when the five-month period after infection was compared to the preceding 7 months. In the 2013 study, the eight herds were missing an average of only 1.0 weaned pig from each litter. Again, this new variant seems to have increased the negative impact on production parameters compared to other PRRSV variants previously seen in Denmark.A reduction in liveborns per litter of between one and two pigs has been reported in several studies after infection with PRRSV –17. SimiInfection with a new recombinant PRRSV-1 variant in 13 herds resulted in a huge decrease in productivity during the subsequent five-month period. Based on the assessment of the data, it can be concluded that the impact of this new PRRSV-1 variant exceeds that previously encountered in Denmark and also exceeds that reported in other countries.Herd owners of herds diagnosed with the new PRRSV-1 variant were contacted via letter and invited to share their production data from 2019. Breeding herds were omitted, since they all completed an elimination programme shortly after infection with the new PRRS1-variant. Only herds that had obtained semen from the infected boar station and sequenced the recombinant of the two PRRSV vaccine strains were included in the study. The identity of the virus was verified by sequencing of samples from individual animals. The sequenced part included at least the full ORF 5 gene and the full ORF 2 gene, but in most cases, the full ORFs 2–7 were sequenced. Only herds in which the virus shared > 99% homology with the Zoetic vaccine strain in ORF 2 and > 99% identity with the Unistrain strain in ORF 5 were regarded as being infected with the recombinant strain and were included in the study. Details on the laboratory methods used have previously been described .To anonymise the inventory, details on the herd size and health status have been omitted from this report.Productivity data from 2018 and 2019 were provided by the herd owners. Since all herds were infected with PRRSV during the month of July, data were divided into the period preceding PRRSV-1 infection and the period after PRRSV-1 infection as follows: preceding PRRSV-1 infection: 01.12.2018–30.06.2019 and after PRRSV-1 infection: 01.08.2019–31.12.2019.Thus, the period “preceding PRRSV-1 infection” covers 7 months, and the period “after PRRSV-1 infection” covers 5 months. The month of July was omitted, since the exact infection time in July could not be confirmed.Farowings/week were calculated as a percentage difference for the preceding 7 months compared to the 5 months after infection. Liveborns/litter, stillborns/litter and weaned pigs/litter were calculated nominally and descriptively as the marginal difference between the preceding 7 months and the 5 months after infection for each of the participating herds. Pre-weaning mortality, including both stillborns and liveborns that died before weaning, was calculated as a percentage for the preceding 7 months compared to the 5 months after infection.Data on the use of PRRS vaccines in 2019 were extracted from VETSTAT . Taking The herd health status before infection with a new PRRSV-1 variant was extracted from the SPF-SuS [


Drosophila melanogaster model to reveal a pivotal role for basement membrane composition in the formation of hematopoietic compartments. We demonstrate that by modulating extracellular matrix components, the fly blood cells known as hemocytes can be relocated to tissue surfaces where they function similarly to their natural hematopoietic environment. We establish that the Collagen XV/XVIII ortholog Multiplexin in the tissue-basement membranes and the phagocytosis receptor Eater on the hemocytes physically interact and are necessary and sufficient to induce immune cell-tissue association. These results highlight the cooperation of Multiplexin and Eater as an integral part of a homing mechanism that specifies and maintains hematopoietic sites in Drosophila.Blood development in multicellular organisms relies on specific tissue microenvironments that nurture hematopoietic precursors and promote their self-renewal, proliferation, and differentiation. The mechanisms driving blood cell homing and their interactions with hematopoietic microenvironments remain poorly understood. Here, we use the  In the animal kingdom, the development and differentiation of immune cells is intimately linked to specific spatial compartments. These sites shelter developing immune precursors from external stimuli while providing signals to orchestrate their self-renewal, proliferation and differentiation. During mammalian embryonic development, the hematopoietic stem cells (HSCs) relocate to the fetal liver where they considerably expand in number, before they populate the spleen and the bone marrow in late embryonic stages . PostnatDrosophila melanogaster emerged as an excellent model to study the dynamics of hematopoiesis  induced a lean phenotype and decreased lipid droplet size  revealed a massive attachment of hemocytes to the adipose tissue surface  domain protein, Activating transcription factor 3 (Atf3), has been defined as a stress-response gene pivotal to the maintenance of immune and metabolic homeostasis , but has4 driver    and the mutation . Since tmutation , we specatocytes .WTHml:DsRed, C7>Atf3 larvae were infected with Leptopilina boulardi parasitoid wasps. We found that the FBAHs detached from the Atf3 overexpressing adipose tissue 48 hr after parasitic infection , mef2-GAL4  and elav-GAL4 (neurons)   drivers neurons) . While l  and overexpressed it with the C7-GAL4 driver in the adipose tissue. Compared to control, Mp levels markedly increased in the basement membrane of C7>Mp::GFP fat body  promoted hemocyte adhesion to this specific domain  agar, 8% cornmeal, 1% soy meal, 1.8% dry yeast, 8% malt extract, and 2.2% sugar beet syrup supplemented with 0.625% propionic acid and 0.15% Methylparaben (Sigma-Aldrich). In standard crosses, 10 virgins were crossed with five males. The crosses were flipped daily, and the larvae were analyzed 6 days after egg laying (AEL). Driver lines crossed to Act5C promoter from the GAL4 coding sequence . Clonal cells were marked by the expression of UAS-GFP or UAS-myr-mRFP. To generate adipocyte clones, first instar larvae (24 hr AEL) were heat-shocked in a 37°C water bath for 20 min, and were afterwards kept at 25°C until processing on day six AEL.Clones overexpressing specific transgene were generated with the FLPout technique utilizing heat shock-induced FLP expression which removes an FRT-flanked stop cassette separating a constitutive Drosophila melanogaster strains were used: 1118w , w; Hml:DsRed , WTw; C7-GAL4, UAS-GFP, Hml:DsRed, UAS-Atf3 (recombined in this study), w; nub-GAL4, UAS-mCherry , +>GAL4, UAS-GFPw; hsFLP, Act5C>y , 1-230, Ubi-mRFP1.NLS.CycB1-266w;; Ubi-GFP.E2f1 , RNAiw; UAS-Mp II , RNAiw; UAS-Col4a1 , RNAiw, UAS-trol , RNAiw; UAS-vkg , RNAiw; UAS-LanA , RNAiw; UAS-LanB1 , RNAiw; UAS-LanB2 , RNAiw; UAS-SPARC , RNAi, UAS-SPARCRNAiw; UAS-Mp (recombined in this study), w; Hml:DsRed; a58-GAL4, UAS-GFP , w; HmlΔ-GAL4, UAS-GFP , w;; UAS-Mp::GFP (this study).The following ml:DsRed , w; C7-G C7-GAL4 , w; C7-GS-Atf3WT , w; C7-G UAS-GFP , w; hsFLS-Atf3WT , w; hsFLycB1-266 , w;; BcFBcF6:GFP , w; Bc1  .Third instar larvae were collected and washed in PBS and placed in glass dissection dishes filled with PBS on ice for 15 min for immobilization. Imaging was performed with an Olympus SZX-16 microscope fitted with a DP72 camera. GFP and RFP images were captured with the cellSens standard v1.11 software  using the Puppet warp tool. Aligned images were projected in Fiji v1.52i (SCR_003070) with ‘Average intensity’ projection, using the ‘Fire’ lookup table to enhance visualization.Stereotypical sessile tissue patterns were generated from larvae imaged under identical conditions. One larva was selected as a reference and every other image was aligned to match the reference in Adobe Photoshop CS5.5  software. Overlapping images were stitched in Fiji v1.52i and all single Z-planes were exported for both GFP (fat body) and DsRed (hemocytes) channels. Each individual confocal section was examined for GFP expression (fat body) and Hml:DsRed expression (hemocyte). The DsRed channel was locally recolored on each section when it was determined to overlap with the GFP signal to amber using Adobe Photoshop CS5.5, then all Z-planes were maximum projected. Sessile- and lymph gland hemocytes are shown in magenta.For confocal imaging of whole fixed larvae, third instar wandering larvae were washed in 70% ethanol and were then injected with 4% paraformaldehyde with a sharpened glass injection capillary. The fixed larvae were immediately placed on a glass slide with a double-sided tape and imaged with an Olympus FV-1000 confocal microscope, with an UPlanSApo 10x (NA0.40) objective. Images were taken using the multi-area module of the FluoView FV-10ASW , anti-Hemese , anti-phospho-histone H3 , anti-Endostatin  antibodies diluted in the blocking solution. After washing, the samples were incubated with the corresponding Cy3- or Cy5-conjugated secondary antibodies (Jackson ImmunoResearch) for 1.5 hr at room temperature and counterstained with DAPI  to visualize nuclei. The fat bodies were dissected from the carcass and mounted in Dabco-Mowiol (Sigma-Aldrich). Tissues were imaged on an Olympus FV-1000 confocal microscope with UPlanSApo 10x (NA0.40), UPlanSApo 20x (NA0.75), UPlanSApo-O 20x (NA0.85), UPlanFLN-O 40x (NA1.30) and UPlanSApo-O 60x (NA1.35) objectives using the FluoView FV-10ASW software. All fat body images represent Z-projections (unless otherwise indicated), which were generated with Fiji v1.52i.Fat body dissection from third instar larvae was performed in PBS by opening the posterior of the larvae and inverting the carcass. After removal of the intestine, larvae were fixed in 4% paraformaldehyde for 1.5 hr. Larval epidermal fillet samples were prepared according to For the pseudocoloring of hemocyte and adipocyte nuclei, confocal Z-stacks of the DAPI channel were divided into surface sections (~0–8 μm from the surface) and deeper sections (>8 μm from the surface), which were projected and exported separately using Fiji v1.52i. The surface projection containing the hemocyte nuclei was then colored amber, while the deeper projection with the adipocyte nuclei was colored magenta. The two projections were merged in Adobe Photoshop CS5.5.L. boulardi wasps for 15 min  on 12 spot glass slides . Allowing hemocytes to adhere for 45 min, samples were then fixed in acetone. Blocking was performed with 0.5% BSA in 1x PBS for 30 min. Hemocytes were stained with anti-Hemese , washed in 100 mM PB and postfixed in 2% osmium tetroxide in PB for 1 hr on ice. Contrasting was performed with 2% uranyl acetate, after which the samples were dehydrated in ethanol and embedded in acetone-resolved araldite. Electron microscopy was performed with an EM 109 (Zeiss) microscope.eater and Mp cDNA was cloned into pENTR4-dual vector between BamHI and NotI sites, and subsequently recombined using LR Clonase II  into pTWG (Mp and Eater) and pTWF (Mp) vectors, respectively . The UAS-Mp::GFP transgenic flies were obtained by standard P-element-mediated germline transformation of pTWG-Mp plasmid into 1118 Drosophilaw embryos . Multiple transformants were recovered and tested, all showing comparable Mp::GFP expression.To express C-terminally GFP- or FLAG-tagged Eater and Multiplexin proteins under UAS control, Drosophila Genomic Resource Center, RRID:CVCL_Z992) were cultured at 25°C in Shields and Sang M3 insect medium  containing 8% fetal bovine serum  without antibiotics. There are no verified reports of Mycoplasma infection in S2 cells . Expression of UAS-driven cDNAs was induced by co-transfection with a pWA-GAL4 plasmid expressing GAL4 under an actin5C promoter. Cells were lysed 36 hr after transfection in lysis buffer containing 50 mM Tris-HCl (pH 7.8), 150 mM NaCl, 1 mM EDTA (pH 8.0), 1% Triton X-100, 0.01% Igepal, and protease inhibitors (Roche Applied Science). Protein concentration was quantified using Bradford reagent  according to manufacturer’s instructions.Schneider 2 (S2) cells (S2 cells . S2 cellAB_2631406) overnight. Following five washes with Lysis buffer, bound proteins were eluted with Glycine-HCl on 37°C, followed by neutralization. Proteins resolved on 10% SDS-PAGE were detected by immunoblotting with anti-Flag M2 , anti-GFP  and anti-α-Tubulin  antibodies, followed by incubation with corresponding HRP-conjugated secondary antibodies (Jackson Immuno Research). Chemiluminescent signal was captured using ImageQuant LAS4000 reader .For each sample, 1 mg of S2 cell protein lysate was incubated with GFP-Trap beads , and equal volumes were resolved on 10% SDS-PAGE. Immunoblotting was carried out with anti-Endostatin , using a customized pipeline. Lamellocyte percentage was determined by comparing the number of L1-positive cells to all counted DAPI-positive nuclei.Fat body associated hemocytes were quantified based on Z-projection images taken with Olympus FV-1000 confocal microscope fitted with UPlanSApo 20x (NA0.75) objective. The area imaged for comparisons is depicted in For the quantification of sessile hemocytes, whole larval images aligned to a reference  were analyzed. These images were loaded in Fiji v1.52i as hyperstacks, containing both control larvae and the genotypes of interest. Using the Rectangle tool, an area was selected where no hemocytes were present, where the average pixel intensity was measured for each control larva and averaged across larvae to establish background levels. Using the Rectangle tool, selections corresponding to single sessile stripes in the A4, A5, A6 and A7 segments were made on control larvae, and the average intensity of the Hml:DsRed signal was measured for the relevant genotypes within this selection. From every measurement the previously established background values were subtracted, then for each larva the four corrected intensity values were added together.SCR_002798), using one-way ANOVA assuming unequal variances with multiple comparisons or unpaired Student's t-test. For comparison of cell counts, one-way Kruskal-Wallis test with Dunn's multiple comparison or nonparametric two-tailed Mann-Whitney test were used. For fat-body-associated hemocytes, every data point represents the number of attached hemocytes within the quantified area, while for circulating hemocytes and sessile hemocyte quantifications, values were normalized to control mean (represented as 1) and shown on the graphs as fold change (F.C.). All experiments represent at least two independent temporal replicates.Statistical analysis and plotting were carried out with Prism 6 .Thank you for submitting your article "Eater cooperates with Multiplexin to drive the formation of hematopoietic compartments" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.eLife". Please let us know if you would like to pursue this option. As the editors have judged that your manuscript is of interest, but as described below that additional experiments are required before it is published, we would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). First, because many researchers have temporarily lost access to the labs, we will give authors as much time as they need to submit revised manuscripts. We are also offering, if you choose, to post the manuscript to bioRxiv  along with this decision letter and a formal designation that the manuscript is "in revision at Summary:In this manuscript, Uhlirova and colleagues have reported an interesting molecular mechanism underlying the hemocyte recruitment to its environments, which bears a clinical potential to human immune cell biology. The authors first focused on a novel phenomenon that overexpression of Atf3 recruits hemocytes to the fat body while altering patterns of hematopoietic pockets in segmentally repeated epidermal sites. The authors went on to understand a molecular basis for the redirection of hemocytes and discovered that Collagen XV/XVIII type protein Multiplexin (Mp) is required for the Atf3-mediated hemocyte homing dependent on the Eater expression in hemocytes. Given that overexpression of Mp in the fat body disrupts the overall pattern of hematopoietic pockets and that Mp is detected in the hemocyte-free hemolymph, the authors raised a valid hypothesis that Mp can be deposited to the hemolymph, which consequently promotes the systemic hemocyte homing to the sessile pockets.Essential revisions:As you will find below, all of the reviewers found the study interesting and agreed on the significance of your work. At the same time, a number of critical criticisms were raised, which require additional data analyses and new experiments. The full comments of the reviewers are attached to provide further details.1) Identify division and differentiation rates of hemocytes in the Atf3 or Mp expression background.- As indicated in the comments below (reviewer 1 and reviewer 2), it will be critical to show the redirection of hemocytes to a new tissue would recapitulate hematopoiesis in the hematopoietic pocket.2) Improve figure images and provide details in figure legends.- There are multiple concerns about this issue from three reviewers. Please find the comments below for details.- Of note, it will be important to adequately address reviewer 3's Essential revisions 3 and 4 where the reviewer raised concerns about images showing the fat body associated hemocytes.3) Provide quantitation of results.- All the reviewers asked for proper quantitations of data for clarity. Please find the details below.Reviewer #1:In this manuscript, Uhlirova and colleagues have reported a critical role for basement membrane proteins in the recruitment of hemocytes in the fat body. The authors first focused on a novel phenomenon that overexpression of Atf3 recruits hemocytes to the fat body while altering patterns of hematopoietic pockets in segmentally repeated epidermal sites. Interestingly, hemocytes attached to the fat body undergo mitosis similar to the ones found in hematopoietic pockets and exhibit normal morphologies. Moreover, hemocytes associated with the fat body relocate to the circulation upon wasp infestation, indicating that these hemocytes display a normal hematopoietic program reminiscent of sessile hemocytes. The authors further went on to understand a molecular basis for the attachment and discovered that Collagen XV/XVIII type protein Multiplexin (Mp) is required for the Atf3-mediated hemocyte homing which is dependent on the Eater expression in hemocytes. Supporting these findings, the authors validated a physical interaction between Mp and eater by biochemical experiments. Given that overexpression of Mp in the fat body disrupts the overall pattern of hematopoietic pockets and that Mp is detected in the hemocyte-free hemolymph, the authors raised a valid hypothesis that Mp can be deposited to the hemolymph critical for the systemic hemocyte homing to the sessile pockets.This study provides a fundamental insight into molecular mechanisms underlying the hematopoietic environment essential for immune cell development and its function and prompts future studies linking the ECM landscape with the hematopoietic pocket formation and hemocyte development.Essential revisions:1) The precise quantitation of data and presenting proper controls will enhance the clarity of the manuscript.1.1) Figure 1C-D': In Figure 1E, the authors quantified FBAH in imaged fat bodies. However, the number of fat body cells per one image could be variable depending on the size of the fat body, dissecting methods or imaging and so on. For example, the mean value shown in Figure 1E is different from Figure 2I even though these data represent the same genotype. It will be important to deliberate on the precise quantitation method for the FBAH phenotype as it is one of the most important findings described in the manuscript.1.2) The control shown in Figure 1E is identical to the one shown in Figure 2I and Figure 3—figure supplement 2E. It will be essential to ALWAYS repeat relative controls side by side with associating experiments.1.3) Figure 2J-K', the number of lamellocytes and the percentage of melanotic capsule formation need to be quantified.1.4) In the same vein, Figure 3—figure supplement 1C-K' requires proper quantitation with enough biological replicates (n) to draw a conclusion.1.5) Figure 1A-B, Figure 3G-H, Figure 4A,G,H, Figure 4—figure supplement 1A-F, Figure 5C-D: authors have shown the relocation of sessile hemocytes. Again, it is one of the main phenotypes shown in the paper; however, none of these are quantitated. The patterns of sessile hemocytes are quite variable even amongst controls. Therefore, authors need to quantify the phenotype with their own measure and with enough sample size.1.6) In Figure 4D-E': though it has been shown in the previous study that eater is essential for the hemocyte homing and that eater is primarily expressed in hemocytes, C7-gal4, hml-gal4  is a different genetic background and can cross-react. Therefore, additional controls  are required at least to be represented as quantitation data.2) It is not clear whether the overall hemocyte number (or hemocyte development) is changed upon Atf3_WT or Mp::GFP expression, which may, in turn, modify the circulating or sessile hemocyte population.2.1) Figure 2C: To claim whether the epidermal sites are more supportive of the proliferation of hemocytes than those in the fat body, the ratio of mitotic cells needs to be quantified as shown in Makhijani et al., 2017. And without showing the number of total hemocytes, it is hard to understand which site is more prominent in the proliferation and differentiation of hemocytes.2.2) The total number of hemocytes together with circulating hemocytes needs to be shown in Atf3_wt or Mp::GFP expression . Moreover, the number of circulating and total hemocytes in Atf3_WT; MpRNAi rescue background should be indicated.3) In Figure 2E-E', it is not clear whether crystal cells express high levels of NimC1 in the images as NimC1-positive membranes are anyway juxtaposed. It will be great to have a bleeding of FABH or high mag of crystal cells with better resolution.4) Though authors have shown that FBAHs are relocated to the circulation upon wasp parasitism, these data are not sufficient to conclude that the fat body FBAH follows the identical hematopoietic program to sessile blood cells. It is possible that FBAHs relocation is less frequent than regular sessile hemocytes found in the epidermis/neuron due to the tight association. It will be important to show whether FBAHs can be reattached after physical disturbances as shown in Makhijani et al., 2011.Reviewer #2:Drosophila and provide convincing evidence that an ECM component, Multiplexin, is necessary for the recruitment of Drosophila immune cells (hemocytes) to the previously identified hematopoietic pockets and for their recruitment to a new position on the fat body where they are can divide and differentiate as they do in the native environment. They define the phagocytosis receptor Eater, which has previously been found to be crucial for hemocyte localization to the hematopoietic pockets, as the partner in mediating hemocyte binding to Multiplexin in both locations. I think this work is exciting and mostly well conducted and deserves to be published in ELife, with some alteration of the text to reflect that some of the current conclusions require a modest amount of new data and analysis.This very well written and interesting manuscript addresses the clinically relevant question of what molecules drive immune cells' interaction with environments that promote their proliferation and differentiation. The authors use the basic system of 1) The paper in places implies that Multiplexin is capable of redirecting hemocytes to a new location in which they can function as in the hematopoietic pocket. This finding would be extremely exciting if true. But though the authors show relocalization to the fat body in the experiment in which they induce clones of Mp , they do not assess the division and differentiation capacity of the hemocytes there. Thus Atf3 expression may induce other changes in the adipocytes than just Mp expression to allow Fat Body associated hemocytes (FBAH) to divide and differentiate there ; Mp may thus be required for hemocyte adhesion yet not sufficient to induce all niche dependent functions. To address sufficiency for proliferation the authors could do a pH3 staining upon the induction of clones in strains they already have . To address sufficiency for differentiation, they could put one copy of Bc1 into that background.Alternatively they should alter the paper to make clearer that they have not explicitly investigated if or shown that the ECM component, Mp, drives hematopoiesis in the Title, the Abstract, the Introduction, the Discussion section .2) They conduct experiments to assess the importance of Mp for attachment to the endogenous sessile niches. Their conclusion (subsection “Similar mechanisms drive sessile hematopoietic pocket formation and FBAH adhesion”) that the knockdown of Mp by RNAi produces a phenotype strikingly resembling that seen in the eater1 mutant  doesn't seem supported by the comparison of Figure panels 4A' to 4H'. While the dorsal patches seem to be gone in the Mp knockdown, it appears as if there is still a repeated hemocyte pattern on the lateral sides in the larvae shown. Having a close up of the lateral and dorsal niches for these genotypes instead of the GFP channel  would clarify this issue. It is clear there is a strongly reduced attachment in the absence of Mp, but if this the same in all the different endogenous hematopoetic locations is important to determine. Given that Mp has been reported to be expressed in the heart tube, next to where the dorsal patches are, and not to my knowledge near the PNS, which flanks the lateral ones, would fit with there being a variable effect in the different regions. This Figure has no quantitation, which also weakens this conclusion. If they do not have the data in hand to make the new figure/ do the quantitation, then they should soften the conclusion that this is the same as the eater1 phenotype and address textually that the effect on the lateral patches has not been rigorously assessed.Reviewer #3:I believe this is a great paper, with exciting science, but I find it unnecessarily difficult to read and to assess critically. I was easily distracted while reading it and I may have missed important points.Essential revisions:1) Conceptually, it would be helpful to give the reasons for choosing to overexpress Atf3 in the fat body. I would also like to see more background information about this transcription factor in the Introduction.2) I also miss a discussion about the possible physiological role of the system described here. Is Atf3 also involved in defining hemocyte docking sites elsewhere? Or should I regard Atf3 overexpression as an artificial but useful experimental system for the study of hematopoiesis?3) In general, the figure legends give little or no information about the experiments shown. Instead they merely describe what conclusions I am expected draw, something that is anyway already explained in the text. There are many examples:a) In Figure 1, the reader has to guess how the different colors are generated. I assume that the green color comes from a UAS-GFP construct, driven by the C7 fat body-specific driver. An Hml:DsRed construct is apparently also involved. It should give red fluorescence to plasmatocytes. Apparently, that this has been converted to the "magenta" color, here labeled "Hemocyte" (I comment the "amber"-colored hemocytes below). Similar difficulties complicate interpretations in other figures too.b) The units on y-axes are sometimes unclear. Does "Number of FBAHs"  refer to the number per unit area as defined in Figure 1—figure supplement 1? And what is "Circulating hemocytes (F.C.)" in Figure 1, Figure 1—figure supplement 1 and Figure 5?c) If "Hemese"  refers to antibody staining, for general visualization of hemocytes, then say so.d) What is "myr-RFP" , and what does it signify?I will stop giving examples here, but my progress through the manuscript was slowed down dramatically by the fact that I constantly had to make guesses about what I could see in the figures.4) How was the "amber"-coloring of hemocytes generated in Figure 1B (and elsewhere)? The text in the Materials and methods section seems to indicate that these cells were simply enhanced in this way via Photoshopping. That does give a striking effect, but is not valid evidence that these cells are attached to the fat body.5) On a similar note, the intracellular vacuoles in Figure 3F are colored "cyan", presumably by Photoshop, to generate the impression that they are contiguous with the similarly colored "pericellular" space under the basement membrane. This is a potentially dangerous way to fool the eye. Do the authors have evidence that these systems are contiguous, or that they are related in other ways? Is there evidence that the associated electron-dense material consists of extracellular matrix proteins?6) As shown here, several extracellular matrix proteins are enriched over Atf3-expressing cells. Does that mean that the basement membrane is thickened? Zooming in on Figure 3F I get an impression that this may be the case.7) I find no information about how the Atf3 clones were generated for Figure 1F, and very rudimentary information about the clones in Figure 3 and Figure 5.8) The magnification and resolution is not sufficient to show the filopodia and lamellipodia in Figure 2A, 2B, Figure 5G and 5H, even when I print out these figures over entire pages. They are even difficult to see when I zoom them in on the computer.9) Has the C7 driver ever been described? The authors refer to a previous article from the same lab , where I only find a reference back to a paper by Grönke et al., (2003). However, C7 is not mentioned there. How specific is the C7 driver, and why is it used?eLife. Your revised article has been evaluated by Utpal Banerjee (Senior Editor) and a Reviewing Editor.Thank you for resubmitting your work entitled "Eater cooperates with Multiplexin to drive the formation of hematopoietic compartments" for further consideration by The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:Your revised manuscript was evaluated by original reviewers (reviewer #1 and reviewer #2) and an additional reviewer (reviewer #4). All the reviewers agreed that now the manuscript is greatly strengthened and is ready for publication after incorporating a few minor changes as suggested by reviewer #2 and #4. Reviewer #4 recommended providing additional statistical information in Materials and methods section and applying generalized linear modeling for count data.Reviewer #1:With an extensive revision, the authors significantly improved the manuscript and satisfactorily addressed all my concerns. Additional data and quantitation looked convincing, and detailed descriptions of the Materials and methods section and Figure legends enhanced its readability.Reviewer #2:The authors have fully addressed my major concerns. I believe the work is greatly strengthened and now makes a convincing case that Mp is capable of inducing the hemocyte proliferation and differentiation in the new fat body location. The quantitation of the Mp knockdown showing the effect on sessile hemocytes has also been conducted, fully anchoring their conclusions on Mp importance for hemocyte localization at endogenous sites of hematopoiesis.I look forward to seeing this exciting work online soon.Reviewer #4:Drosophila blood cells, the hemocytes, with other tissues during the formation of sessile hemocytes clusters. The manuscript provides evidence on a key role of Multiplexin in the formation of these clusters, which, as this manuscript and previous work shows, are important sites for hemocyte proliferation and probably also differentiation. The reviewers in the first round of assessment have pointed out valid concerns regarding the manuscript and, in my opinion, the authors have done a good job addressing these concerns. I have only few remaining points/questions for the authors.This manuscript provides novel insight into the interaction of Drosophila material is not exactly scarce. However, I don't see this as a decisive problem in this study, one reason being that differences between controls and experimental crosses are in general quite clear. Also, multiple experiments conducted back each other up. However, could authors provide more detailed information on how experiments were replicated? Specifically, were the animals studied from the progeny of one cross/the same patch of crosses or were experiments replicated temporally? This could be clarified in the Materials and methods section since it helps in assessing the reproducibility of the results.On some occasions, the number of animals tested seem quite low, especially when  Reviewer #1:In this manuscript, Uhlirova and colleagues have reported a critical role for basement membrane proteins in the recruitment of hemocytes in the fat body. The authors first focused on a novel phenomenon that overexpression of Atf3 recruits hemocytes to the fat body while altering patterns of hematopoietic pockets in segmentally repeated epidermal sites. Interestingly, hemocytes attached to the fat body undergo mitosis similar to the ones found in hematopoietic pockets and exhibit normal morphologies. Moreover, hemocytes associated with the fat body relocate to the circulation upon wasp infestation, indicating that these hemocytes display a normal hematopoietic program reminiscent of sessile hemocytes. The authors further went on to understand a molecular basis for the attachment and discovered that Collagen XV/XVIII type protein Multiplexin (Mp) is required for the Atf3-mediated hemocyte homing which is dependent on the Eater expression in hemocytes. Supporting these findings, the authors validated a physical interaction between Mp and eater by biochemical experiments. Given that overexpression of Mp in the fat body disrupts the overall pattern of hematopoietic pockets and that Mp is detected in the hemocyte-free hemolymph, the authors raised a valid hypothesis that Mp can be deposited to the hemolymph critical for the systemic hemocyte homing to the sessile pockets.This study provides a fundamental insight into molecular mechanisms underlying the hematopoietic environment essential for immune cell development and its function and prompts future studies linking the ECM landscape with the hematopoietic pocket formation and hemocyte development.Essential revisions:1) The precise quantitation of data and presenting proper controls will enhance the clarity of the manuscript.1.1) Figure 1C-D': In Figure 1E, the authors quantified FBAH in imaged fat bodies. However, the number of fat body cells per one image could be variable depending on the size of the fat body, dissecting methods or imaging and so on. For example, the mean value shown in Figure 1E is different from Figure 2I even though these data represent the same genotype. It will be important to deliberate on the precise quantitation method for the FBAH phenotype as it is one of the most important findings described in the manuscript.SPARC or vkg, the overall size of the adipose tissue was not noticeably affected by co-expression of additional transgenes with UAS-Atf3WT.We expanded the Materials and methods section to accurately describe how the quantifications were performed. The area of the fat body assessed throughout the study and across the different genotypes was specifically selected because of its flat surface and absence of additional tissues that can associate with this organ . Furthermore, while structural variances could indeed be observed in response to manipulating various genes, such as Regarding the differences in FBAHs numbers in Figure 1E and Figure 2I, we consider different food composition as an important and likely source of variability in the number of FBAHs between the two datasets mentioned. The wasp infection experiments were performed in the lab of Dr. Tomas Dolezal . This means that flies/larvae were reared on different fly food than the larvae throughout the manuscript. The numbers therefore should be interpreted within the context of the particular experiment. We now describe in detail the experimental setup for the wasp infestation experiments in the Materials and methods section including the fly food composition.1.2) The control shown in Figure 1E is identical to the one shown in Figure 2I and Figure 3—figure supplement 2E. It will be essential to ALWAYS repeat relative controls side by side with associating experiments.The control numbers (indicated in gray) were repeated because these genotypes were dissected and quantified as parts of the same experiments to allow comparison across genotypes. To align with the order of the data presentation, the data had to be “artificially” split. We now provide additional data with separate controls for each experiment shown. In the case of Figure 3K and Figure 3—figure supplement 1I we indicate in the supporting datasets that the results belong to the same experiment.1.3) Figure 2J-K', the number of lamellocytes and the percentage of melanotic capsule formation need to be quantified.C7>Atf3WT larvae, and we did not intend to draw conclusions about the efficiency of the immune response. We adjusted the wording of the manuscript to indicate this.We now include the quantification of lamellocytes . The capsule formation rate cannot be assessed as the wasp infestation experiments were performed in the lab of Dr. Tomas Dolezal  that is currently not accessible to us due to the travel ban for the University of Cologne employees due to COVID-19 pandemic. We would like to note that the data shown in Figure 2J-O was only included to indicate that lamellocyte differentiation and encapsulation is not noticeably affected in 1.4) In the same vein, Figure 3—figure supplement 1C-K' requires proper quantitation with enough biological replicates (n) to draw a conclusion.We performed the quantifications which are now included in Figure 3—figure supplement 1I and reflect on the data in the text.1.5) Figure 1A-B, Figure 3G-H, Figure 4A,G,H, Figure 4—figure supplement 1A-F, Figure 5C-D: authors have shown the relocation of sessile hemocytes. Again, it is one of the main phenotypes shown in the paper; however, none of these are quantitated. The patterns of sessile hemocytes are quite variable even amongst controls. Therefore, authors need to quantify the phenotype with their own measure and with enough sample size.We devised a quantification method of the sessile hemocyte patterns based on image analysis. The description of the quantification method is now included in the Materials and methods section. The results are included in the relevant figures. For data shown in Figure 4—figure supplement 1A-F, we adapted relevant statements in the Results section.1.6) In Figure 4D-E': though it has been shown in the previous study that eater is essential for the hemocyte homing and that eater is primarily expressed in hemocytes, C7-gal4, hml-gal4  is a different genetic background and can cross-react. Therefore, additional controls  are required at least to be represented as quantitation data.C7>Hml>Atf3WT and C7>Hml>Atf3WTeaterRNAi and included the results in the manuscript and in Figure 4H-J.We performed quantifications comparing 2) It is not clear whether the overall hemocyte number (or hemocyte development) is changed upon Atf3_WT or Mp::GFP expression, which may, in turn, modify the circulating or sessile hemocyte population.2.1) Figure 2C: To claim whether the epidermal sites are more supportive of the proliferation of hemocytes than those in the fat body, the ratio of mitotic cells needs to be quantified as shown in Makhijani et al., 2017. And without showing the number of total hemocytes, it is hard to understand which site is more prominent in the proliferation and differentiation of hemocytes.The manuscript does not state that “the epidermal sites are more supportive of the proliferation of hemocytes than those in the fat body”, only that the epidermally associated hemocytes proliferate more than freely circulating ones, citing the data and conclusions from Makhijani et al., 2017. Our intention was to determine the behavior of hemocytes on the fat body surface, and the phenomena of hemocyte proliferation and plasmatocyte-crystal cell trans-differentiation are indicative of an unchallenged, homeostatic hemocyte developmental program.Furthermore, we indicate in the Discussion section that differences likely exist between FBAHs and sessile hemocytes, but the mechanistic basis of their attachment is conserved.2.2) The total number of hemocytes together with circulating hemocytes needs to be shown in Atf3_wt or Mp::GFP expression . Moreover, the number of circulating and total hemocytes in Atf3_WT; MpRNAi rescue background should be indicated.Atf3WT and MpRNAi expression in the fat body were aimed to show that on one hand the hemocyte attachment to the fat body is dependent on Mp, and on the other hand, when hemocytes cannot attach to the adipose tissue the pattern of the sessile hematopoietic compartment is restored, for which we now include additional data in Figure 3L-O. We would also like to note that the comparison of total hemocyte numbers using the methods from Petraki et al., 2015 may not reflect the accurate values, as those are optimized for circulating and sessile cells, and not hemocytes attached to the internal organs.The experiments combining 3) In Figure 2E-E', it is not clear whether crystal cells express high levels of NimC1 in the images as NimC1-positive membranes are anyway juxtaposed. It will be great to have a bleeding of FABH or high mag of crystal cells with better resolution.We exchanged the panels in Figure 2E-E’ to better show the colocalization of the Bc:GFP signal and the NimC1 staining on the intermediate hemocytes.4) Though authors have shown that FBAHs are relocated to the circulation upon wasp parasitism, these data are not sufficient to conclude that the fat body FBAH follows the identical hematopoietic program to sessile blood cells. It is possible that FBAHs relocation is less frequent than regular sessile hemocytes found in the epidermis/neuron due to the tight association. It will be important to show whether FBAHs can be reattached after physical disturbances as shown in Makhijani et al., 2011.We did not intend to suggest that FBAHs and sessile hemocytes behave “identically”, rather that tissue/fat bodyattached hemocytes seem to behave in a similar fashion. To reflect this more accurately, we changed the wording of the manuscript. On a second note, similarly to Essential revisions 2.2, the physical mobilization assay described in Makhijani et al., 2011 was specifically created to release sessile hemocytes which adhere to the epidermis and are sensitive to external force . FBAHs on the other hand are on the surface of the adipose tissue, which is floating in the hemolymph, and are therefore much less prone to these insults.Reviewer #2:Drosophila and provide convincing evidence that an ECM component, Multiplexin, is necessary for the recruitment of Drosophila immune cells (hemocytes) to the previously identified hematopoietic pockets and for their recruitment to a new position on the fat body where they are can divide and differentiate as they do in the native environment. They define the phagocytosis receptor Eater, which has previously been found to be crucial for hemocyte localization to the hematopoietic pockets, as the partner in mediating hemocyte binding to Multiplexin in both locations. I think this work is exciting and mostly well conducted and deserves to be published in ELife, with some alteration of the text to reflect that some of the current conclusions require a modest amount of new data and analysis.This very well written and interesting manuscript addresses the clinically relevant question of what molecules drive immune cells' interaction with environments that promote their proliferation and differentiation. The authors use the basic system of 1) The paper in places implies that Multiplexin is capable of redirecting hemocytes to a new location in which they can function as in the hematopoietic pocket. This finding would be extremely exciting if true. But though the authors show relocalization to the fat body in the experiment in which they induce clones of Mp , they do not assess the division and differentiation capacity of the hemocytes there. Thus Atf3 expression may induce other changes in the adipocytes than just Mp expression to allow Fat Body associated hemocytes (FBAH) to divide and differentiate there ; Mp may thus be required for hemocyte adhesion yet not sufficient to induce all niche dependent functions. To address sufficiency for proliferation the authors could do a pH3 staining upon the induction of clones in strains they already have . To address sufficiency for differentiation, they could put one copy of Bc1 into that background.Alternatively they should alter the paper to make clearer that they have not explicitly investigated if or shown that the ECM component, Mp, drives hematopoiesis in the Title, the Abstract, the Introduction, the Discussion section .We now include experiments demonstrating the cell proliferation as well the presence of crystal cells among hemocytes attached to Mp::GFP overexpressing fat body clones . We would like to add that while hemocyte division does take place in the clusters attached to the Mp::GFP clonal cells, the amount of attached hemocytes and the size of the clones vary among experimental replicates making the quantification of these phenomena problematic.Furthermore, we include new data showing that Mp::GFP overexpression in the wing imaginal disc, also causes the specific attachment of hemocytes, further strengthening the evidence for the crucial role of Mp to facilitate hemocyte-tissue association . Additionally, we reworded the cited conclusions to more appropriately describe the phenotypes observed.2) They conduct experiments to assess the importance of Mp for attachment to the endogenous sessile niches. Their conclusion (subsection “Similar mechanisms drive sessile hematopoietic pocket formation and FBAH adhesion”) that the knockdown of Mp by RNAi produces a phenotype strikingly resembling that seen in the eater1 mutant  doesn't seem supported by the comparison of Figure panels 4A' to 4H'. While the dorsal patches seem to be gone in the Mp knockdown, it appears as if there is still a repeated hemocyte pattern on the lateral sides in the larvae shown. Having a close up of the lateral and dorsal niches for these genotypes instead of the GFP channel  would clarify this issue. It is clear there is a strongly reduced attachment in the absence of Mp, but if this the same in all the different endogenous hematopoetic locations is important to determine. Given that Mp has been reported to be expressed in the heart tube, next to where the dorsal patches are, and not to my knowledge near the PNS, which flanks the lateral ones, would fit with there being a variable effect in the different regions. This Figure has no quantitation, which also weakens this conclusion. If they do not have the data in hand to make the new figure/ do the quantitation, then they should soften the conclusion that this is the same as the eater1 phenotype and address textually that the effect on the lateral patches has not been rigorously assessed.The experiments showing the disruption of the sessile compartment are now complemented with additional images, showing closeups of the dorsal and lateral patches . Additionally, to provide better representation for the sessile tissue pattern, we now include quantifications for the relevant genotypes, and compare the stereotypical sessile tissue pattern with larvae where the compartment is disrupted . We would like to note that on the overview images, it is possible to interpret the hemocytes as “repeated hemocyte pattern”, but in reality, this is due to the immobilization (cooling the larvae) and the settling of the circulating hemocytes. Since the contractions of the dorsal vessel diminish in low temperature, the cells in the circulation can accumulate between the muscle and epidermal layers, creating the impression of a repeated pattern .Reviewer #3:I believe this is a great paper, with exciting science, but I find it unnecessarily difficult to read and to assess critically. I was easily distracted while reading it and I may have missed important points.Essential revisions:1) Conceptually, it would be helpful to give the reasons for choosing to overexpress Atf3 in the fat body. I would also like to see more background information about this transcription factor in the Introduction.See below.2) I also miss a discussion about the possible physiological role of the system described here. Is Atf3 also involved in defining hemocyte docking sites elsewhere? Or should I regard Atf3 overexpression as an artificial but useful experimental system for the study of hematopoiesis?Drosophila tissues in an accessible manner. As the reviewer suggested, we now include additional background about Atf3 in the leading paragraph of the Result section.The use of Atf3 was based on our previous observation of the hemocyte attachment to the fat body. The overexpression of Atf3 in the fat body at high levels is indeed artificial, and our goal was to exploit this as a platform to understand the fundamental mechanisms underlying hemocyte attachment to 3) In general, the figure legends give little or no information about the experiments shown. Instead they merely describe what conclusions I am expected draw, something that is anyway already explained in the text. There are many examples:a) In Figure 1, the reader has to guess how the different colors are generated. I assume that the green color comes from a UAS-GFP construct, driven by the C7 fat body-specific driver. An Hml:DsRed construct is apparently also involved. It should give red fluorescence to plasmatocytes. Apparently, that this has been converted to the "magenta" color, here labeled "Hemocyte" (I comment the "amber"-colored hemocytes below). Similar difficulties complicate interpretations in other figures too.b) The units on y-axes are sometimes unclear. Does "Number of FBAHs"  refer to the number per unit area as defined in Figure 1—figure supplement 1? And what is "Circulating hemocytes (F.C.)" in Figure 1, Figure 1—figure supplement 1 and Figure 5?c) If "Hemese"  refers to antibody staining, for general visualization of hemocytes, then say so.d) What is "myr-RFP" , and what does it signify?I will stop giving examples here, but my progress through the manuscript was slowed down dramatically by the fact that I constantly had to make guesses about what I could see in the figures.We expanded the Figure legends and the Materials and methods section considerably to facilitate reading, including the specific suggestions of the reviewer.4) How was the "amber"-coloring of hemocytes generated in Figure 1B (and elsewhere)? The text in the Materials and methods section seems to indicate that these cells were simply enhanced in this way via Photoshopping. That does give a striking effect, but is not valid evidence that these cells are attached to the fat body.We added the description to the Materials and methods section. Briefly, larvae were fixed with the injection of 4% paraformaldehyde, immobilized on microscope slides and image with confocal microscope. Each individual confocal section was examined for GFP expression (fat body) and Hml:DsRed expression (hemocyte). The DsRed channel was locally recolored on each section when it was determined to overlap with the GFP signal. The attachment of the hemocytes is also demonstrated throughout the study on dissected fat bodies .5) On a similar note, the intracellular vacuoles in Figure 3F are colored "cyan", presumably by Photoshop, to generate the impression that they are contiguous with the similarly colored "pericellular" space under the basement membrane. This is a potentially dangerous way to fool the eye. Do the authors have evidence that these systems are contiguous, or that they are related in other ways? Is there evidence that the associated electron-dense material consists of extracellular matrix proteins?We based our assessment on two previous publications, which used similar indication for the pericellular spaces, and concluded that the trapped material consists of ECM proteins. The representative Figure from Zang et al., 2015, shows the same type of distinction between pericellular spaces and cytoplasm/organelles, and points out the electron dense material as ECM protein aggregates.6) As shown here, several extracellular matrix proteins are enriched over Atf3-expressing cells. Does that mean that the basement membrane is thickened? Zooming in on Figure 3F I get an impression that this may be the case.C7>Atf3WT fat bodies. In controls the pericellular space between the cell membrane and the BM is considerably wider.We include a closeup image 7) I find no information about how the Atf3 clones were generated for Figure 1F, and very rudimentary information about the clones in Figure 3 and Figure 5.We generated the clones using the hsFLPout system. We now include a description in the Materials and methods section and add description into respective Figure legends.8) The magnification and resolution is not sufficient to show the filopodia and lamellipodia in Figure 2A, 2B, Figure 5G and 5H, even when I print out these figures over entire pages. They are even difficult to see when I zoom them in on the computer.We now include higher magnification images of these samples with the filopodia and lamellipodia indicated.9) Has the C7 driver ever been described? The authors refer to a previous article from the same lab , where I only find a reference back to a paper by Grönke et al., (2003). However, C7 is not mentioned there. How specific is the C7 driver, and why is it used?C7-GAL4 is a strong fat body-specific driver expressed in the fat body cells from early larval stages. A detailed description of the driver can be found in Koyama and Mirth, 2016, which we now refer to in the manuscript.The Reviewer #4:Drosophila blood cells, the hemocytes, with other tissues during the formation of sessile hemocytes clusters. The manuscript provides evidence on a key role of Multiplexin in the formation of these clusters, which, as this manuscript and previous work shows, are important sites for hemocyte proliferation and probably also differentiation. The reviewers in the first round of assessment have pointed out valid concerns regarding the manuscript and, in my opinion, the authors have done a good job addressing these concerns. I have only few remaining points/questions for the authors.This manuscript provides novel insight into the interaction of Drosophila material is not exactly scarce. However, I don't see this as a decisive problem in this study, one reason being that differences between controls and experimental crosses are in general quite clear. Also, multiple experiments conducted back each other up. However, could authors provide more detailed information on how experiments were replicated? Specifically, were the animals studied from the progeny of one cross/the same patch of crosses or were experiments replicated temporally? This could be clarified in the Materials and methods section since it helps in assessing the reproducibility of the results.On some occasions, the number of animals tested seem quite low, especially when All experiments represent at least two independent temporal replicates. We now include the sentence in Materials and methods section.


Molecular analysis techniques such as gene expression analysis and proteomics have contributed greatly to our understanding of cancer heterogeneity. In prior studies, gene expression analysis was shown to stratify patient outcome on the basis of tumor-microenvironment associated genes. A specific gene expression profile, referred to as ECM3 (Extracellular Matrix Cluster 3), indicated poorer survival in patients with grade III tumors. In this work, we aimed to visualize the downstream effects of this gene expression profile onto the tissue, thus providing a spatial context to altered gene expression profiles. Using infrared spectroscopic imaging, we identified spectral patterns specific to the ECM3 gene expression profile, achieving a high spectral classification performance of 0.87 as measured by the area under the curve of the receiver operating characteristic curve. On a patient level, we correctly identified 20 out of 22 ECM3 group patients and 19 out of 20 non-ECM3 group patients by using this spectroscopic imaging-based classifier. By comparing pixels that were identified as ECM3 or non-ECM3 with H&E and IHC images, we were also able to observe an association between tissue morphology and the gene expression clusters, showing the ability of our method to capture broad outcome associated features from infrared images. Although cancer has been thought as a consequence of genetic mutations in an aberrant tissue mass, tumor progression is increasingly viewed as functionally interconnected with the surrounding microenvironment2. Indeed, crosstalk between cancer cells and the tumor microenvironment components  plays a major role in regulating several biological processes3. Differences in ECM-associated gene expression can describe the biological and clinical heterogeneity of breast cancer, especially in terms of prognosis and response to therapy2. Recent works have identified a subgroup of breast cancers characterized by overexpression of a robust cluster of genes mainly encoding structural ECM proteins, originally referred to as the ECM3 group5. These features indicate an epithelial to mesenchymal transition (EMT) but with accelerated metastatic potential only in the undifferentiated (grade III) phenotype5. Notably, ECM3 classification provides additional information to assess tumor progression5. It also identifies tumors in which the interplay between myeloid-derived suppressor cells (MDSCs) and the ECM drives the induction of EMT in tumor cells, and suggests a strategy for inhibition of MDSC by aminobisphosphonates that could revert EMT and lead to less aggressive tumor phenotype6.Breast cancer is the most prevalent cancer in womenEven though such molecular classification is of great importance in stratifying patients, it requires multi-step tissue processing and still requires the validation of an algorithm for classification at the single tumor level to be used instead of a clustering analysis on gene expression data of a cohort of samples. Additional information that could confirm this phenotype, without requiring multiple complicated steps or affecting the molecular integrity of the tissue, would be impactful. We thus sought a technology that could provide a molecular characterization of the stroma and provide this information in the context of the tumor to inform decision-making. Typical imaging techniques that could provide this context require labeling or other workflows that may not be compatible with established methods of genetic analyses of ECM3.11. It can be used to probe biomolecular changes in the tumor microenvironment12 for comprehensive spatio-molecular assessment of the tissue which can be useful for determining prognosis13. We hypothesized that the differences in gene expression profiles between ECM3 and non-ECM3 tumors would also result in broad molecular composition changes in the tissue, which can be read with FT-IR spectroscopic imaging. Further, by associating gene expression profile with tissue molecular profiles, we can begin observing where on the tissue the downstream effects are the most prominent, thus spatially mapping the effect of altered gene expression.In this work, we evaluate an emerging technique for combined spatial and molecular analysis, infrared spectroscopic imaging, to map the downstream effects of this stromal gene expression onto the tissue. We then use this spectral information to determine outcome-associated patient groups. Fourier transform infrared (FT-IR) spectroscopic imaging is effective in providing spatially specific molecular analysis of samples for pathology, without the need for stains or dyes5. In this work, we performed FT-IR spectroscopic imaging of 42 breast carcinomas, of which 22 were ECM3 and 20 were non-ECM3 as evaluated by gene expression profile analysis. Our goal was to find IR patterns that discriminate ECM3 from non-ECM3 tumors  were observed among samples, even when collected over several months . The slope of the linear fit model (0.13 ± 0.02) was significantly different from zero at p-value 8.9 e-7, indicating linear relationship between gene expression data and the IR imaging-based analysis , IR scores for both T1 and T2 appear to be within a similar range , including several collagen chains (16 genes). Accordingly, a specific IR spectral pattern in the collagen area associated with the ECM3 gene expression profile. To our knowledge, this is the first indication that IR can visualize the product of altered gene expression in tissues. Considering that ECM3 and non-ECM3 tumors are classified based on the expression of ECM genes, an all-optical method to detect the same could be very useful. Given that there are differences in collagen spectra in the protein-rich region of the IR spectrum, it seems reasonable to expect that protein backbone signatures in IR could provide this discrimination. Indeed, most of the ECM3 genes (43/58) encode structural ECM proteins involved in the maintenance of connective tissue , other than proteolytic enzymes. IR imaging is one of the few analytical methods able to provide information about collagen biodistribution and assemblyIn this work, we identified a spectral predictor of ECM3 tumors in the collagen areas and the IR model explained 44% of the variability observed by gene expression. The gene expression analysis was carried out with frozen tissue samples, notably, while the IR imaging analysis was carried out with paraffin-embedded tissue sections. In addition, the areas used for both the techniques were adjacent but different, with the tissue needing to be crushed for gene expression analysis. Thus, we do expect some variability to arise from both processing as well as differences between the tissue sections. However, the concordance between the two is very exciting. Their observed relationship supports the ability of this technique in capturing the physiologic consequences of altered gene expression on the tissue, despite the substantial differences in the techniques that were used to probe the sample, as well as differences in sample preparation protocol.32, in addition to conventional methods of measuring protein expression with IHC, there are no techniques to visualize the effects of a group of genes interacting together. From mapping the heterogeneity derived from the overexpression of the ECM-related genes, we observed that the stroma in which the ECM3 tumor is developing is different from non-ECM3 tumors at biochemical or structural organization levels.We also utilized the spatio-biomolecular information obtained with IR spectroscopic imaging to visualize the biological effect derived from the altered gene expression in the tissue. By comparing the classified images to H&E and Masson’s trichrome images, we were able to see a correspondence between the changes in the collagen in the tumor microenvironment and the ECM classification by IR spectra. While it has recently been made possible to spatially map specific gene expression by amplifying the gene in small fragments of tissue31. While most of IR absorptions are very similar between collagen types, a combination of signals from 4 spectral intervals  was demonstrated to generate a robust classifier of collagen type31. These data suggest that a different ratio of collagen types in the area of collagen fibrils could be the basis of the ECM3 classifier. Collagen VI amount in the TME could likely be relevant for ECM3 classification by FT-IR. This indication is reasonable considering that type VI collagen presents the most evident spectral difference among collagen types31 and that ECM3 signature is characterized by overexpression of genes transcribing the three chain of collagen VI 5 In addition, ECM3 tumors express higher level of collagen VI by immunohistochemistry than non-ECM3 tumors5. It is also possible that ECM3 FT-IR signals identify areas with differences in collagen organization. IR spectra for collagens in the region we analyzed (872–1320) are mainly due to hydroxyproline, proline and phenylalanine residues other than structural α helix and amide III residues33. These residues are relevant for collagen elasticity and stiffness34, indicating possible roles in collagen organization. This association is also likely considering the differences in the collagen organization we observed in ECM3 pixels compared to non-ECM3. The different collagen organization is likely to derive from overexpression of different fibrillar collagen chains  together with collagen VI, a highly disulfide cross-linked microfibrillar collagen described to modify the organization of the fibrillar collagens intertwining with them35.Further analyses are needed to dissect the specific molecules or chemical/biological modification that explain the observed IR spectrum. However, it is conceivable that differences between ECM3 and non-ECM3 tumors are derived from the presence of different collagen types and/or in their organization in the microenvironment. Indeed, IR analysis has been previously described by several groups to differentiate the presence of different collagen types by their secondary structure parameters36 differently. It is also possible that collagen organization derived from the interaction between undifferentiated tumor cells (grade III) and ECM3 TME is behind tumor progression, as observed in general in breast cancer37, and that this peculiar organization is not readable with IR imaging in the collagen areas. Further studies are needed to dissect the molecular features of the ECM3 TME that are responsible for patients’ poor prognosis.Despite the clear distinction between the ECM3 and non-ECM3 groups, the primary cause of the poor prognosis of patients with ECM3 grade III tumors remains unexplained. In this work, we did not find differences in collagen spectra based on the tumor grade. It is possible that collagen organization is not different in ECM3 grade III compared to ECM3 grade I-II tumors but that undifferentiated (grade III) tumor cells sense the TME, as the collagen VI, reported to favor tumor growth and progression2. However, the speed of acquisition can be improved by manifolds with the recent advancements in high-definition38 and high-speed41 IR spectroscopic imaging coupled with machine learning to perform image analysis and prediction. In addition, classification rendered by IR spectroscopic imaging is less processing-intensive, providing prediction in a much simpler workflow than the conventional gene expression analysis protocols. Although confirmatory studies on independent case series are needed to evaluate the reproducibility of this classifier, the optimal cut-off and its prognostic performance in interaction with tumor grade, this methodology may have direct implications for future clinical assessment on breast cancer patients.In conclusion, we were able to identify an optical spectral ECM3 classifier as a relatively easy method to identify ECM3 tumors. As a proof of concept, the IR-spectroscopic imaging method utilized here is based on conventional Fourier Transform infrared spectroscopic imaging instruments, requiring 10–24 hours of acquisition for every surgical resection between 1–5 cm42. Data of ECM classification, analyzed by using the Large Average Submatricies (LAS) biclustering algorithm described in Triulzi et al.5 were retrieved from the study. Expression data are available in the Gene Expression Omnibus data repository (GEO) with accession number GSE59595.The 42 tumors analyzed in this study were selected among a cohort of 97 consecutive primary breast cancer patients treated at Fondazione IRCCS Istituto Nazionale dei Tumori of Milan  and characterized through whole gene expression profile analysis on HumanHT-12-v3 expression Bead Chip by IlluminaAll procedures were per the Helsinki Declaration. Biospecimens used for research consisted of leftover material of samples collected during standard surgical and medical approaches at INT. Samples were donated by patients to the Fondazione IRCCS Istituto Nazionale dei Tumori BioBank for research purposes, and aliquots were allocated to this study after approval by the Institutional Review Board and a specific request to the Independent Ethical Committee of the institutes. The use of tissue for this study was approved by the University of Illinois Institutional Review Board via project 06684.Formalin-fixed paraffin-embedded (FFPE) slides sequential to that used for FT-IR analysis were stained with H&E and with Masson’s Trichrome Stain Kit (Agilent) for direct visualization of collagen fibers. Stained slides were digitized by a slide scanner .−1 spectral resolution and 6.25 µm pixel size.5 µm thick FFPE breast cancer samples were sectioned onto IR-transparent Barium Fluoride salt plates. Sections were deparaffinated by washing in xylene for 24 hours, changing the solvent every three hours. FT-IR spectroscopic images of the tissue sections were collected using a Perkin Elmer Spotlight 400 system, equipped with 16 elements linear focal plane array HgCdTe detector. For each pixel, four scans were averaged at 4 cm43. Images were stitched from the image tiles, baseline corrected, and normalized. The infrared images were converted to spectral metrics by calculating peak heights, areas and locations and taking ratios of these variables. Using these metrics, a previously developed breast supervised classifier was applied to the IR data to identify areas classified as collagen28.Image processing was performed in ENVI-IDL 4.8 with in-built and in-house written algorithms−1 and 1826 cm−1 bands and compared by student’s t-test. While it appears that there is no significant difference between the SNR of the groups, due to low power we added a spectral smoothening step by application of Savitzky-Golay 9-point filter to further ensure that SNR did not affect our analysis. Spectral data points between 872 cm−1 and 1320 cm−1 were used for training. Data were cleaned by removing out of range observations with absorbance values greater than 2 or less than −1. For the training set, the mean collagen spectra per patient were calculated. Principal components analysis was performed to reduce the data dimensionality, and the first 20 components were chosen for further study. To perform principal components analysis, we used the raw intensity values at each spectral band collected between 872 cm−1 and 1320 cm−1. Linear discriminant classification with 5-fold cross-validation was used to develop the ECM3 classifier to classify ECM3 and non-ECM3 spectra. Hyperparameters that minimized the 5-fold cross-validation loss were automatically determined by using Matlab’s automatic hyperparameter optimization protocols. From this, the optimal linear coefficient threshold Delta was determined to be 0.0001674 at 0.003 regularization parameter value. Delta threshold was used to determine the eligibility of variables for inclusion in the model, while the regularization parameter was used to prevent overfitting. Post ECM3 classification, the classifier was projected onto the full IR images and the total number of pixels classified as ECM3 and non-ECM3 were calculated. The percentage of ECM3 pixels out of the total number of collagen pixels was used to stratify patient groups. These values were fit in a logistic regression model to identify the optimal cutoff point for patient stratification.Post identification of collagen areas, all further data analysis was performed in Matlab R2016a. Baseline corrected and normalized infrared spectrum was extracted from areas identified as collagen in the histology classifier. To ensure that the data collected over several months did not have significant deviations in terms of signal to noise ratio (SNR), SNR was calculated as the ratio of peak intensities of 1656 cmFisher’s exact test tested association among categorical variables. Two-sided p < 0.05 was considered significant. Survival functions were assessed using the Kaplan-Meier estimator, while the log-rank test was used to compare survival distributions.Supplementary information.