Cancer divergence has many facets other than being considered a genetic term. It is a tremendous challenge to understand the metastasis and therapy response in cancer biology; however, it postulates the opportunity to explore the possible mechanism in the surrounding tumor environment. Most deadly solid malignancies are distinctly characterized by their tumor microenvironment (TME). TME consists of stromal components such as immune, inflammatory, endothelial, adipocytes, and fibroblast cells. Cancer stem cells (CSCs) or cancer stem-like cells are a small sub-set of the population within cancer cells believed to be a responsible player in the self-renewal, metastasis, and therapy response of cancer cells. The correlation between TME and CSCs remains an enigma in understanding the events of metastasis and therapy resistance in cancer biology. Recent evidence suggests that TME dictates the CSCs maintenance to arbitrate cancer progression and metastasis. The immune, inflammatory, endothelial, adipocyte, and fibroblast cells in the TME release growth factors, cytokines, chemokines, microRNAs, and exosomes that provide cues for the gain and maintenance of CSC features. These intricate cross-talks are fueled to evolve into aggressive, invasive, migratory phenotypes for cancer development. In this review, we have abridged the recent developments in the role of the TME factors in CSC maintenance and how these events influence the transition of tumor progression to further translate into metastasis and therapy resistance in cancer. TM. TM33]. ΔIEC colitis mice model. Low levels of IFN γ-induced stemness in a dose-dependent manner to activate the ICAM-PI3K-Akt-Notch1 axis in non-small cell lung cancer [Interferons (IFNs) are cytokines that regulate various biological activities such as immune response, cell proliferation, and viral infections. It is classified into three major types. Among type1  and type II (IFN γ), and type 3 are all presented in mammals . The inng cancer . Also, ag cancer .Tumor necrosis factor α (TNF-α), a pleiotropic cytokine, is one of the major elements regulating the TME and plays a vital role in inflammation, immunity, apoptosis, angiogenesis, stemness, and cancer progression . It cont+ positive population in lung cancer cells [+ CSC-like cells in response to the radiation treatment of lung cancers [+ innate immune cells secreting IL-6, which favors stemness and enhances adaptive inflammatory response to assist tumor growth in colorectal cancer [Interleukin 6 (IL-6) is released by macrophages, T cells, and fibroblasts in the TME and maintains a pro-tumor environment on other cells in the TME to encourage tumor evasion and angiogenesis. It interacts with cancer cells via various downstream mediators to promote cancer growth, and their signaling has been shown in various mice and human models . IL-6 pler cells . IL-6 in cancers  and prom cancers . IL-6 re cancers . Ovarian cancers . Colonicl cancer . IL-6 mol cancer .Targeting IL-6 as a therapeutic approach to overcome chemoresistance in tumor formation is critical, and HIF is required for cancer cell chemoresistance. IL-6 increased the chemoresistance of ovarian cancer cells in vivo and in vitro, according to a recent study, by upregulating HIF-1 and STAT3 signaling . BMI1 is+IL-17+ T cells possess the ability to promote tumor-initiating cells. This research reveals that co-culturing spherical cells with Foxp3 + IL-17 + T cells dramatically increased the stemness markers CD133, CD44s, CD166, EpCAM, and ALDH1. Further, they validated this effect using a neutralizing anti‑IL‑17 antibody and observed that the stemness effect was abolished after the antibody treatment. Further, Xiang et al. [+ cancer stem-like cells. Ovarian CD133+ cancer stem-like cells significantly increased sphere formation ability by IL-17 overexpression, which might be mediated by NFκB and p38/MAPK signaling pathway. Another study showed that IL-17E binding to IL-17RB activates the JAK/STAT3 and NFκB pathways to promote stemness in human hepatocellular carcinoma cells. This effect is revoked by using JAK and NFκB inhibitors [IL17 is another important cytokine derived from TME. IL-17 is secreted as a disulfide-linked homo and hetero dimer peptide with six members  . Yang etg et al.  showed thibitors . IL17 anhibitors . Furtherhibitors  showed thibitors . Furtherhibitors . These s+ stem-like cells promote regulatory T cells and inhibit T cell proliferation compared to CD44 stem cells [MDSCs are vital components in the TME, promoting cancer growth, stemness, and metastasis. MDSCs recognize other immune cells to produce cytokines and execute immunosuppressive functions. MDSCs act as a major immune response regulator and a key target for cancer treatment . A studyem cells . Furtherem cells . MDSCs eem cells . Anotherem cells . Ai et aem cells . Recentlem cells . Overallin vivo model. Further, lung cancer cells co-cultured with M2 TAMs significantly increased mucin protein MUC1 and stemness gene expression. MUC1-silenced M2-TAMs showed fewer stemness features and concluded that TAMs play an essential role in lung cancer progression [IFN-(lipopolysaccharide) classically activates M1 macrophages, causing them to produce cytokines and initiate the immune response. M2 macrophages are alternatively activated by cytokines IL-4, IL-10, and IL-13 to produce the polyamine or proline to induce proliferation and collagen production in wound healing . These Mgression . Huang egression  found thFibroblasts are a common stromal cell type found in the connective tissue and produce the extracellular matrix and collagen to maintain a structural framework of the tissues. It has a branched cytoplasm containing a speckled nucleus with two or more nucleoli. Fibroblasts secrete precursors of ECM to support a structural framework on connective tissue and play a significant role in the wound healing process . In normThe trans-differentiated myofibroblasts further received cues from different sources to activate and accrue heterogeneity to maintain the expression of several markers . These a+/GPR77+ CAFs have been identified to be a subtype of CAFs found in breast cancer patients to promote stemness and drug resistance by cleaving C5a, a protein fragment produced by complement component cleavage [CAFs are a major source of extracellular matrix synthesis in the tumor stroma. They deliver a wide range of soluble factors that aid and maintain tumor progression, angiogenesis, and the CSC niche and promote drug resistance . Giannoncleavage . Furthercleavage , ovariancleavage , and luncleavage . We rececleavage . CAFs alcleavage . So, CAFEndothelial cells are specialized cells forming the linings of blood vessels, creating a life-supporting system for migrating cells in every part of the body. Further, endothelial cells can remodel and extend the blood vessel network and tissue growth. Endothelial cells containing the capillaries related to arteries and veins in the vascular system create the vascular bed of the whole body. This vascular system is required for normal physiological functions such as organ development and tissue regeneration. So, tumor vasculature was established through a critical process known as neo-angiogenesis for tumor survival and expansion. RNA sequence experiments elucidated the distinct lineages of endothelial cells in TME. These subgroups of endothelial cells promoted the cancer progression from the non-invasive to the invasive stage of tumor development . FurtherLu et al.  showed tAdipocytes are specialized energy-storing fat cells primarily composed of adipose tissue in the whole body. Adipocytes are white and brown adipocytes in which white adipocytes secrete hormones, adipokines such as resistin, adiponectin, leptin, and brown adipocytes provide to generate heat for the body . Normal Several studies have shown that adipose-derived stem cells play a role in cancer progression and enrich the stemness in cancer cells. Multipotent adipose-derived stem cells can differentiate into osteocytes, myocytes, and neuron-like cells. Adipose-derived stem cells are characterized based on their CD13, CD31, CD34, CD45, CD73, CD90, and CD105 markers expressions on their surface . Goto et+ CSC marker expression in the fusion cell population. Interestingly, they found fusion efficiency was high in CD44-enriched MDA-MB-231 breast cancer cells with adipose-derived stem cells fused hybrid cells. The knockdown of CD44 significantly reduced cell fusion efficiency. According to the findings of this study, CD44-mediated cell fusion is critical in developing CSC in breast cancer cells [+ CD24− tumor-initiating cells in MCF-7 and MDA-MB-231 cells [+/CD24−, MDR1+ and PDL1+ populations in triple-negative breast cancer cells and declined the in vivo tumor growth. When combined with other standard treatments, secretome formulation was found to be a viable biotherapeutic option for breast cancer patients [Adipose-derived stem cells in the TME could influence the therapeutic sensitivity of various cancer. Condition media collected from human adipose-derived stem cells promoted doxorubicin resistance in triple-negative breast cancer cells . Adiposeer cells . Adipose31 cells . A recenpatients . It was TME plays a vital role in protecting cancer cells from drug action in all solid human malignancies. TME mediates their activity through cross-talk with CSC and other TME cellular components to develop therapy resistance . CAFs, tCAFs have a high tumorigenic potential and can further mediate drug resistance and the proliferation of therapy-resistant cells following drug treatment. Additionally, TME plays a crucial role mediate therapy resistance. So, the chemotherapeutic drugs created a non-targeted effect on CAFs in the TME. CAF-released exosomes with long non-coding RNAs improved cisplatin chemoresistance in small lung cancer cells . CAFs-deATP binding cassette (ABC) transporter are the largest family members of transmembrane proteins, with 49 members and seven gene sub families labeled ABCA-G and found in all living organisms. These proteins transport substrate across the intracellular membrane using ATP hydrolysis-mediated energy utilization and have a wide range of physiological functions, including detoxification, oxidative stress defense, xenobiotics, lipid metabolism, antigen presentation, and stem cell drug resistance . These AThe apoptosis mechanism is activated via two pathways: extrinsic and intrinsic. The extrinsic pathway is initiated by various death receptors , and the intrinsic pathway is mediated by mitochondria and releases apoptotic factors (cytochrome C). A group of cysteine proteases enzyme “caspases” carried out both pathways . One of + CSC population in lung cancer cells promoted radioresistance through altered regulation of DNA repair genes. Replication stress, a form of DNA damage, is pivotal in therapeutic resistance in CSCs. A recent study showed breast cancer CSCs have less replication stress than non-breast cancer CSCs. The breast cancer CSCs have the BMI1 protein placed around stalled replication forks to engage RAD51 to activate the homologous recombination machinery to confirm that the BMI1/RAD51 axis is necessary to prevent cisplatin-induced cisplatin DNA damage [DNA Damage Response (DDR) is an endogenous cell DNA lesions machinery to repair lesions and maintain genomic stability. TME and CSCs have a capacity-efficient DNA DDR system that protects the cancer cells from DNA-damaging therapeutic agents. Platinum-based therapeutic drugs and radiotherapy eradicated cancer cells using DNA damage. Bao et al.  showed tA damage . This stThe stemness-related pathways, such as hedgehog, Wnt/β-catenin, and Hippo pathways, were involved in the therapeutic failure of all solid malignancies . LodestiColorectal cancer patients are highly suitable for chemotherapy and frequently relapse into the tumor. Stromal factors are responsible for contributing to the chemoresistance of colorectal cancer. Microenvironment components HIF-1α and CAF-derived TGF-β2 promoted the hedgehog transcription factor GLI2 in CSCs, enhancing intrinsic resistance to drug therapy . Human oWnt/β-catenin is a highly conserved pathway that regulates cellular events, including proliferation, migration, apoptosis, and stem cell maintenance. This pathway has been identified as an important target for TME-CSC-based therapy resistance. In pancreatic cancer, stromal pancreatic stellate cells secreted Wnt and tenascin C (TnC) ligand molecules promoting the β-catenin and YAP/TAZ signaling pathways. Furthermore, N-Myc downstream-regulated 1(NDRG1) and DNA protein cross-link (DpC) reduced cross-talk between pancreatic cancer cells and pancreatic stellate cells by inhibiting β-catenin and YAP/TAZ nuclear localization and promoting DKK1, a Wnt inhibitor, in pancreatic cancer cells . MesenchHippo pathways play an essential role in TME-CSC-mediated chemoresistance in solid malignancies. Hippo pathways have transcriptional co-activators such as YAP and TAZ that play an essential role in embryonic development, proliferation, cancer progression, CSC maintenance, and therapy resistance . This paCancer metastasis is a life-threatening event in all solid malignancies, and 90% of cancer patient mortality happens at a later stage of the disease. The failure to manage the disease continually raises cancer-related death each year. Metastasis is a multidimensional evolution of fundamental biological processes in which cancer cells move from their originating site to form a tumor colony in distant organs and tissues. Intravasation, survival, and diffusion into the circulation, extravasation, and the ability of mesenchymal cells to transform into epithelial cells, resulting in colonization and micrometastasis at distant secondary sites . In secomilieu. These cells may reside in original tumors, originate through metastatic cascade events, or develop the ability to engage with microenvironmental components such as immunological, endothelial, and stromal cells after arriving at a distant site [Metastasis-initiating cells boost cellular plasticity and stemness by hijacking stem cell pathways, yet, these cells can also survive the metastatic cascade in a specific organ or tissue ant site . The ori+ CSCs with strong pro-invasive and pro-metastatic potential blocked ENO1 expression and downregulated caveolin-1 expression, both of which are necessary for invadopodia formation and CSC invasiveness. In gastric cancer, peritoneal metastatic cancer stem cells (pMCSCs) promote invasiveness and partial EMT in establishing peritoneal tumors. Furthermore, these created pMCSCs consistently expressed CD44 and CD54 markers, which are important in the gastric cancer metastasis. TGF adaptor protein-2 spectrin (SPTBN1) is a critical protein in liver regeneration and acts as a tumor suppressor, and its downregulation has been shown frequently in hepatocellular carcinoma [In the metastasis beginning site, a tiny subset of metastatic cancer stem cells is capable of invasive and self-renewal capability. Metastatic cancer stem cells have represented the CSC hierarchy with suitable molecular markers in the metastasis region. α-enolase (ENO1), a glycolytic enzyme produced on the surface of tumor cells, has been identified as a key metastasis-promoting factor. ENO1 was recently discovered in the invadopodia surface of the human stomach and prostate adenocarcinomas by Huang et al. . They alarcinoma . From anarcinoma .+ metastatic cancer stem cell population. The unique abilities to switch between cancer stem cells and metastatic stem cells to initiate tumors for appropriate tissue microenvironment instead of host immune surveillance were discovered in this work [HIF-1 and HIF-2 were found to govern metastasis-initiating cells and their progenies during hypoxic cancer progression. The tumor survival and invasion of metastasis-initiating cells are influenced by activated hypoxia factors that regulate pluripotency-associated transcription factors, EMT, and angiogenic indicators . The rolhis work . So, theMetastasis-initiating cells have the ability with stem-like and immune-evasive properties. Recent research shows that tumor cell seeding to secondary sites occurs early in the tumor and can be as dormant as single cells or micrometastases. The surrounding tumor microenvironments govern metastatic dormancy and interact with metastatic progeny stem-like cells to trigger colony development in distant sites . UnderstIn the secondary sites, myeloid-derived suppressor cells had a pleiotropic function in shaping the metastatic tumor microenvironment, allowing tumor cells to escape the innate and adaptive immune response . MDSCs cTo cross the endothelium during intravasation and extravasation, cancer cells require the disruption of endothelial junctions, known as transendothelial migration. Tumor cells cause angiogenesis, and new blood vessels weaken the cell-cell connection, allowing them to easily enter the vasculature during the intravasate . VEGF anCSCs were selected based on their surface maker expression to target the therapeutic strategy to eliminate the risk of recurrence in the developmental stage of all human cancers . The theTME plays a vital role in regulating stem-like events to maintain the plasticity in cancer cells. So, targeting cross-talk factors between cancer cells and TME may prove powerful approach than targeting CSC alone. CSCs could acquire drug resistance by interacting with TME components. Thus, targeting the TME components may signify an effective therapeutic strategy for TME-mediated stemness enrichment and prevent therapy resistance. CAFs represent a major component of TME and play an imperative role in therapy resistance. Thus, targeting the CAFs may be a critical therapeutic approach to improving clinical outcomes. FAP protein was expressed on the surface of CAF cells. However, recent studies showed that FAP is expressed in bone marrow stem cells. Systemic anti-FAP therapy may be toxic side effects on cells. As a result, nanoparticle-based photoimmunotherapy (nano-PIT) was used to eliminate CAFs in tumor-bearing immunocompetent mice, and this approach also increased T cell infiltration and tumor suppression . RecentlImmunotherapy approaches to blocking antibodies such as CTLA-4, PD-1, or PD-L1 might benefit advanced cancer patients. Further combination with other checkpoints or therapeutics molecules improved the immune response. PD-1 therapy response was significantly improved using a combination with CSF surface-modified bladder cancer stem cells vaccine . FurtherCSCs appeared in a heterogeneous population of tumor cells. Therefore, the identification of CSCs remains challenging. Screening, identification, and use of multiple markers that provide specific tumor types and drug responses. Combination therapy may be more efficient than targeting single molecules or pathways. Immunotherapy is a potential option for targeting CSCs with checkpoint molecules; however, CSCs are less immunogenic than non-CSCs and thus have a limited anti-tumor response.Organoids are an important model for preserving tumor heterogeneity and drug resistance in high-throughput drug screening. Circulating tumor cells are another promising model because they represent genetic and epigenetic characteristics within the tumor and are a popular drug-testing model. Metastatic stem cell interacts with the local and metastatic microenvironment, and updated knowledge about the cellular and molecular mechanisms governing the animal models of metastatic stem cells should shed light on the potential therapeutic application in the TME- mediated CSC role in metastasis.Transcriptional heterogeneity in TME and cancer cells have undeviating clinical implications. To elucidate the molecular classification of a single biopsy might prevent the unbiased way to imply clinical management; however, single-cell RNA sequence experiments demonstrated the presence of multiple cell populations in various molecular groups within the tumor and elucidated the mutual cross-talk in symbiotic diverse cell populations in the TME.Targeting CSCs and TME may represent a wise choice; however, the interconnected network and cues are complex and obscure. TME components have different types of cells, cytokines, and growth factors. CSCs have proved to be targeted to improve cancer patients; however, therapeutics of TME-mediated stemness is still challenging. The focus of future clinical trials in TME/CSC may focus on combination therapy to target multiple pathways and careful consideration of reducing the toxicity to normal cells, improving the drug selectivity and efficacy, and exploring the possibility of the mode of delivery. In conclusion, combining TME/CSC targeted therapeutic molecules with conventional chemotherapeutic drugs may provide a better direction for anti-cancer therapy, resulting in improved cancer patient survival rates and reduced recurrence.


Chimeric antigen receptor (CAR) T-cell therapy, belonging to adoptive immune cells therapy, utilizes engineered immunoreceptors to enhance tumor-specific killing. By now new generations of CAR T-cell therapies dramatically promote the effectiveness and robustness in leukemia cases. However, only a few CAR T-cell therapies gain FDA approval till now, which are applied to hematologic cancers. Targeting solid tumors through CAR T-cell therapies still faces many problems, such as tumor heterogeneity, antigen loss, infiltration inability and immunosuppressive micro-environment. Recent advances provide new insights about the mechanisms of CAR T-cell therapy resistance and give rise to potential reversal therapies. In this review, we mainly introduce existing barriers when treating solid tumors with CAR T-cells and discuss the methods to overcome these challenges. Immune therapies are powerful for cancer treatment and, regardless of the drug types used , tumor regression usually involves participation of the immune system , 2. Immund CAR T-cell therapies since 2017 as the result of positive clinical cases for anti-blood cancer treatment including acute lymphoblastic leukemia (ALL), non-Hodgkin lymphoma (NHL), follicular lymphoma, mantle cell lymphoma (MCL) and multiple myeloma  patient in 2013, which indicated the anti-solid tumor efficacy of engineered T-cell therapies . CAR-engin vitro and in vivo suggesting it may be a promising target for epithelial cancers  and tumor associated fibroblasts and plenty of them are cold tumors, which means they lack complete blood vessels and proper conditions to recruit immune cells –57 Fig. CXCR1- + T-cells recruitment  secreted by MDSCs recruits Th17 cells which strengthen the immunosuppressive effect. Treg activation strongly inhibits CD8+ cell proliferation and it is reported that deleted the IL1R1+ Treg could improve T-cell ability in solid tumors  are now well studied and heavily heterogeneous. Most ECM is the product of CAFs including immunosuppressive fibroblast activation protein (FAP). Deletion of these cells provides opportunities for immune therapies . In partd tumors . Tumor ceraction . Thus, Teraction . FRβ is eraction . This steraction , 110. Aleraction . One the through creening . Deletio T-cells . Thus, P T-cells , 161. It T-cells . However T-cells . And one T-cells .Though some combined therapies may be suitable for both hematological malignancy and solid tumors, we should be careful about the intrinsic differences between them. IFN-gamma signaling is well known for its importance to control tumor growth and mutation of IFN-gamma related component can result in resistance of immune therapies . One groAfter many years’ attempt, many researchers finally turn the concept of CAR T-cells into a powerful adoptive cell therapy and it acquired so much success in the past few years especially for blood malignancy. Advances learned from hematologic tumors treatment equip CAR T-cells with new-generation armors but there are still lots of considerations due to the complexity of the solid tumor itself, the microenvironment and immune system. Luckily, more and more fantastic ideas are placed into practice including CAR modifications, T-cell enhancement and collaborative therapies. These methods provided positive cases in many solid tumor models and even in patients. Beside the evolution on CAR T-cells, the differences between blood and solid tumors are getting clear and discovery of pan-immune system modulators such as PTPN family members strengthens the application of CAR T therapies. In short, CAR T-cells have great potential to be easily modified according to rising concepts to reverse specific tumor resistance. However, the remaining problem is how to optimize CAR T-cells through integrating the advantages of existing and new-born methods, which needs more research practice and clinical investigations.On the other hand, few studies discuss the challenges of CAR T therapy during the CAR manufacture process and the component of CAR T-cells may also play an important role during solid tumor treatment such as the ratio of different T-cells. Furthermore, CAR T therapies induced side effect is a barrier restricting the effective cell dose during solid tumor treatment. Larger cell dose means both stronger opposite and side effect, so it is also valuable to explore the proper methods to reduce off-tumor effect. In all, CAR T-cells treating solid tumors are promising with the continuous efforts on the bench and in the hospital.GX and YQ conceptualized the frame work. YQ wrote the manuscript and made the figures under the supervision of GX. YQ and GX contributed to the editing of the content. All authors reviewed the article and agreed to the submitted version.


Rapastinel, formerly Glyx-13, is a novel positive allosteric modulator of the N-methyl-D-aspartate-receptor (NMDAR) that counteracts psychotomimetic actions of NMDAR antagonists. We set out to evaluate the effect of rapastinel alone or in combination with the global and GluN2B subunit–specific NMDAR antagonists MK-801 and Ro25-6981, respectively, on neuronal activation in relevant regions using c-fos brain mapping. Whereas rapastinel alone did not trigger significant c-fos expression beyond the prelimbic cortex, it strongly increased the c-fos expression induced by MK-801 in hippocampal, cingulate, and retrosplenial areas. Similar results were obtained when rapastinel was replaced by D-cycloserine. Our results reveal new interactions at network level between NMDAR modulators with possible implications regarding their therapeutic effects. Glutamatergic agents are increasingly studied as a possible treatment avenue in depression and schizophrenia, and recently esketamine has been approved as an adjunctive therapy in treatment-resistant depression . HoweverWe aimed to evaluate the network effects of rapastinel, focusing on brain regions involved in depression and psychosis. Using c-fos mapping, we determined the effect of rapastinel alone or in combination with the global and GluN2B subunit-specific NMDAR antagonists MK-801 and Ro25-6981, respectively. We also compared the effect of rapastinel on c-fos expression with that of D-Cycloserine, an NMDAR modulator binding at the glycine site .Experiments were performed on 2 groups of adult C57BL6 mice (n = 84) purchased from Charles River, Sulzfeld, Germany as breeding pairs, as previously described . The firSections were incubated free-floating with anti-c-fos antibodies . Staining was visualized using nickel-3,3′-diaminobenzidine . Cell coP < .05 considered statistically significant.Statistical analyses were performed using the statistical program SPSS 23 for Windows. The mean number of c-fos–expressing cells was calculated for each treatment group, and differences between groups were evaluated by use of 1-way ANOVA followed by post hoc Bonferroni testing, with The c-fos brain mapping revealed a clear difference between the activation patterns of the different glutamatergic agents used. Rapastinel induced a significant c-fos expression in the prelimbic but not infralimbic area of the medial prefrontal cortex . In accoRapastinel alone did not induce a significant c-fos expression beyond the prelimbic cortex, as previously reported . HoweverOur results illustrate an unexpected potential of rapastinel and D-cycloserine to activate, possibly by disinhibition, large cortical networks when added to MK-801. While such allosteric modulators alone disinhibit pyramidal neurons in select brain regions , this efPresent results may have implications for understanding the role of glutamatergic agents in treating psychiatric disorders. Previous data indicated a role of the GluN2B subunit in mediating the antidepressant effect of both rapastinel and ketamine . We founOur data identify for the first time, to our knowledge, complex interactions between different NMDAR modulators at the network level. Further functional studies may unravel the mechanisms underlying these effects and the potential therapeutic benefit or disadvantage of their combination.


Cutaneous neurosensory symptoms have become increasingly reported findings in COVID-19; however, these virus-related manifestations are largely overlooked, and their pathology is poorly understood. Moreover, alterations of skin sensibility currently recognize no clear histopathology substrate. The purpose of this study was to provide pathology evidence of neurosensory skin system involvement in COVID-19 patients complaining of subjective neurological symptoms affecting the skin. Out of 142 patients, six long COVID-19 cases complaining of cutaneous subjective neurological symptoms assessed on an NTSS-6 questionnaire underwent histopathological and immunohistochemical analyses of skin areas affected by paroxysmal diffuse burning and itching sensations. Two patients also performed electroneurography examination. The histology investigation showed hypertrophic glomus vascular bodies with hypertrophic S100+ perineural sheath cells and adjacent hypertrophy of the nerve branches associated with increased basophil polysaccharide matrix. Electroneurography revealed disturbances of A-delta and C dermal neuronal fibers. The main limitation of this study consisted of a limited number of skin biopsy samples, requiring further investigation. Histopathology findings are consistent with hypertrophy of nerve endings, suggesting a condition such as “dermal hyperneury”, a recently reported small nerve hypertrophy condition affecting sensory C fibers. Such a neuropathic basis could explain dysesthesia experienced by the patients, as previously described in postherpetic neuralgia. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) typically manifests with interstitial pneumonia or with acute respiratory distress syndrome (ARDS) and multisystem involvement in severe cases. It is now established that SARS-CoV-2 is able to infect a wide variety of cells, including neurons, by exploiting the angiotensin-converting enzyme 2 (ACE2) entry receptor expressed almost ubiquitously in human tissues . The corTo this date, the etiology underlying COVID-19 neuropathies remains poorly understood, and histopathology is still lacking.The purpose of this study was to investigate the cutaneous pathology underlying sNS of largely overlooked burning and itching skin sensations in long-COVID-19 patients. Histology and immunohistochemistry studies were performed on skin samples from each subject.Out of 142 subjects who consulted our post-COVID-19 follow-up clinic (PCFC) of the Dermatology department in “Sapienza” University of Rome from May to September 2021, we enrolled six COVID-19 cases complaining of cutaneous sNS following SARS-CoV-2 infection . All patFurthermore, skin biopsies of the burning involved areas were effectuated to perform histology and immunohistochemistry. Finally, all patients underwent follow up clinical visits after 3 and 6 six months.A 49-year-old Caucasian woman consulted our PCFC complaining of cutaneous burning pain, arising bilaterally on the shoulders, extending on the lateral surface of both arms, and gradually becoming generalized. The skin examination showed red-purple erythema on the palmar region, bilaterally, coexistent with livedo reticularis on the legs. Livedoid skin manifestations appeared two weeks earlier and rapidly improved in the following days. Though the patient presented with bilateral erythema of the palms, the clinical suspect of erythromelalgia was excluded due to the absence of typical triggers for episodic pain and to the widespread burning sensation involving apparently normal distant skin. The positive serum antibody test against SARS-CoV-2 confirmed the diagnosis. The rheumatological and autoimmune vasculitis panel was negative as well. The histology examination of the right palmar skin showed glomus vascular bodies, hypertrophic S100+ peri-neural sheath cells, and adjacent hypertrophy of the nerve branches with increased basophil polysaccharide matrix . No inflA 67-year-old Caucasian woman was referred to the PCFC following recovery. The patient complained of burning pain and an itching sensation, beginning in the late period of COVID-19 infection. Skin pain started on the chest, extending irregularly to the four limbs and reaching the palmar and plantar surfaces. The skin examination was negative. A biopsy was performed on the skin with burning pain in the abdominal area. Histology findings showed small nerve ramifications in the medium and deep dermis. Fibers appeared wavy and hypertrophic and displayed a slight increase in basophil mucopolysaccharide content. Intraepidermal S100+ dendritic cells were irregularly distributed . CD34+ iA 64-year-old Caucasian man was referred to our PCFC in December 2020 following the onset of a generalized cutaneous sensation of burning and itching skin. The patient contracted the SARS-CoV-2 infection 2 months before and was admitted to our hospital for pulmonary involvement with characteristic anosmia and dysgeusia. Acute symptoms resolved in 2 weeks, leaving asthenia, shortness of breath, and mental fog, indicating a long-COVID-19 condition. Dermatology was consulted for the management of the skin burning—paresthesia. The skin biopsy was performed on the skin in the abdominal region. Histology showed hypertrophic nerve bundles that were markedly visible in the mid dermis . AdditioA 35-year-old Caucasian woman presented to our PCFC complaining of the onset of hives in December 2020, following recovery from a COVID-19 pericarditis requiring hospitalization in September. The patient reported an intense persistent itching and stinging sensation. The skin biopsy was obtained from popliteal itchy skin. A glomeruloid cluster of nerve bundles with basophilic mucin surrounding the nerve sheath was present in the mid dermis without an inflammatory reaction . Other sA 62-year-old Caucasian man consulted our PCFC complaining of paresthesia and dysesthesia in the skin of the right hemithorax and the medial surface of the right lower limb. The patient developed severe COVID-19 pneumonia in November 2021, requiring admission to the intensive care unit. The personal history was otherwise uneventful, except for arterial hypertension. ENG showed reduced sensory nerve action potential amplitude consistent with sensory axonal neuropathy; nerve conduction velocity was normal. The histology examination revealed epidermal hyperkeratosis, acanthosis, and disappearance of rete ridges. Perivascular inflammation, characterized by plasma cells, eosinophils, and lymphomonocytic infiltrate, was also observed . S100-stA 50-year-old female patient complained of acute hair loss initiating 1–3 months after the onset of SARS-CoV-2 infection. The patient reported intense trichodynia before and during the hair shedding. The trichoscopy showed typical telogen effluvium patterns. The trichogram showed a telogen rate greater than 20%. The pull test result was positive. A 4-mm punch biopsy of the scalp was performed. Histology showed hair follicles in anagen, telogen, and catagen phases. Median anagen/telogen ratio was 50%. No inflammatory infiltrate was present. There were various stages of dystrophic changes with evidence of keratin condensation as an initial event, followed by bulb atrophy up to fibrosis. The presence of hypertrophic nerve trunks revealed the hallmark of a hyper-neuritis condition .The patient was prescribed daily oral supplementation with alpha-lipoic acid, biotin, calcium pantothenate, ferrous gluconate, and vitamin D3 for 3 months.Long COVID-19 syndrome is relatively common, affecting from 20 to 65% of patients after the acute phase of the disease. It is characterized by the persistence of symptoms such as fatigue, shortness of breath, and brain fog that cannot be explained by alternative diagnosis and that are not correlated with the severity of acute COVID-19. Peripheral neuropathy and neurosensory alterations are rarely reported in this condition. Neurologic involvement during SARS-CoV-2 infection may concern both central and peripheral nervous systems, being that the ACE2 receptor is widely expressed across neuronal cells ,8,9,10. Many pathogenetic mechanisms have been hypothesized for COVID-19 peripheral nervous system involvement. The immune-mediated effect of anti-GD1b autoantibodies and para-infectious damage of the peripheral nerves were described in Guillain-Barré and Miller-Fisher syndrome. Additional patho-mechanisms could be related to both the direct effect of SARS-CoV-2 or the indirect damage during the “cytokine storm”. It was also reported that SARS-CoV-2 may potentially access the nervous system and spread by anterograde and other routes of axonal transport, causing complex perceptive alterations ,12,13.Peripheral nervous system injury in the course of COVID-19 is likely underrecognized, and the incidence of its associated symptoms is likely to be underestimated, with reports ranging from 2.3%  to 5.8% Our study investigated cutaneous sNS in subjects with acute COVID-19, as well as with long COVID-19 syndrome, integrating clinical examination with histopathologic analysis and immunohistochemistry. Out of 142 patients examined, nine subjects 8.28%) complained of subjective symptoms compatible with neurosensory cutaneous involvement; six patients consented to skin biopsy. A shared feature in these patients was the subjective perception of burning pain, which was generalized over the cutaneous surface, did not follow specific dermatomeric distribution, and was apparently unrelated to objective skin lesions. The skin pain occurred in the late phases of COVID-19, and women were more frequently affected in line with the literature [.28% compParesthesia, alterations of pain and temperature sensation, are established sensory neurologic symptoms related to SARS-CoV-2 infection that may be due to an indirect mechanism related to virus-induced immune dysregulation ,23. SimiHistopathology and immunohistochemistry in our study showed dermal nerve fiber hypertrophy manifesting as readily visible nerve bundles in the reticular dermis. This finding is typically described in prurigo nodularis due to increased Calcitonin gene-related peptide (CGRP)  and in tThe neuropathic chronic pain in post-acute COVID-19 patients has been recently described by Ieremia et al., who reported loss of dermal neurosensory fibers in nine patients . CompareParoxysmal attacks of burning and itching sensation related to COVID-19, and Long-COVID-19 as well, can be related to the damage of the neurosensory skin system, manifesting with uneven distribution, affecting the skin in a skip fashion irrespective of dermatomes. The symptoms have variable onsets in the course of SARS-CoV-2 infection and may persist for months, possibly accompanied by systemic manifestations such as asthenia, shortness of breath, and brain fog.Histopathologic findings showed different alterations of small dermal fibers, depending on disease duration. Hypertrophy and inflammation may occur in the early phase of the disease, followed by atrophy and fiber loss in the chronic stage. Our experience in the management of COVID-19-related sensory neuropathic pain suggests that this condition is scarcely responsive to oral antihistamines. We have obtained satisfactory responses in our patients with low-dose oral gabapentin administered for three months. At a follow up visit after 6 months, all subjects reported remission of symptoms.


To evaluate the feasibility of a novel approach for predicting hepatocellular carcinoma (HCC) response to drug-eluting beads transarterial chemoembolization (DEB-TACE) using computed tomography hepatic arteriography enhancement mapping (CTHA-EM) method.TPost/pre-RE) defined as the ratio of tumor enhancement to normal liver tissue was calculated to categorize tumor response as residual (TPost-RE > 1) versus non-residual (TPost-RE ≤ 1) enhancement, which was blinded compared to the response assessment on first follow-up imaging using modified RECIST criteria. Additionally, for tumors with residual enhancement, CTHA-EM was evaluated to identify its potential feeding arteries.This three-institution retrospective study included 29 patients with 46 HCCs treated with DEB-TACE between 2017 and 2020. Pre- and posttreatment CTHA-EM images were generated using a prototype deformable registration and subtraction software. Relative tumor enhancement . The first follow-up imaging showed non-complete response  in 19/46 (41.3%) and complete response in 27/46 (58.7%) HCCs. CTHA-EM had a response prediction sensitivity of 94.7%  and specificity of 100% . Feeding arteries to the residual enhancement areas were demonstrated in all 18 HCCs .CTHA-EM showed residual enhancement in 18/46 (39.1%) and non-residual enhancement in 28/46 (60.9%) HCCs, with significant differences on CTHA-EM method was highly accurate in predicting initial HCC response to DEB-TACE and identifying feeding arteries to the areas of residual arterial enhancement.The online version contains supplementary material available at 10.1186/s41747-022-00315-8. Computed tomography hepatic arteriography enhancement mapping (CTHA-EM) correlated with initial hepatocellular carcinoma response to drug-eluting beads transarterial chemoembolization (DEB-TACE).CTHA-EM provided quantitative evaluation of residual arterial tumor enhancement after DEB-TACE.CTHA-EM identified feeding arteries to areas of residual tumor enhancement.CTHA-EM might be used to guide treatment delivery and determine treatment endpoint.Transarterial chemoembolization (TACE) is a standard treatment for patients with intermediate-stage hepatocellular carcinoma (HCC) , 2. Its In lipiodol-based TACE, the degree of intratumoral lipiodol accumulation can be utilized as a surrogate for treatment response and can be assessed by intraprocedural computed tomography (CT) or cone-beam CT (CBCT) –12. In dAdvanced algorithms that apply deep learning methods on DSA image sequence are being developed to predict treatment response . HoweverCT hepatic arteriography (CTHA) has proved to be superior to CBCT for TACE planning because of its better contrast resolution, comparable spatial resolution, and minimal artifacts caused by motion and beam hardening . CTHA alThe aim of this study was to evaluate the feasibility of a novel approach to predict HCC response to DEB-TACE using CTHA enhancement mapping (CTHA-EM) through image subtraction.n = 33 patients), (3) who were deemed to have achieved complete response (CR) by intra-procedural DSA and CT (n = 30 patients), and (4) who had at least one follow-up CT or magnetic resonance imaging following DEB-TACE  treatment-naïve HCC without extrahepatic arterial tumor supply, (2) who were treated with DEB-TACE and had a dual-phase CTHA images  acquired before and after DEB-TACE delivery  loaded with doxorubicin  or epirubicin  and mixed with 12, 15, or 19 mL of nonionic contrast medium and 6, 5, or 10 mL of 0.9% saline, respectively. The solution was injected manually at a rate of approximately 1 mL/min until complete tumor devascularization and near stasis of the feeding artery was documented on DSA images. All DEB-TACE were performed with the goal of achieving CR per modified response evaluation criteria in solid tumors (mRECIST)  –29 26–29ROITumor — largest tumor cross-sectional area as seen on either axial or coronal plane; ROINormal — normal liver parenchyma in the contralateral hepatic lobe, excluding blood vessels) and transferred to posttreatment CTHA-EM image. Tumor relative enhancement on pre-(TPre-RE) and post-(TPost-RE) DEB-TACE CTHA-EM images was calculated as the ratio of HU values of tumor enhancement to the normal non-embolized liver parenchyma . Tumor response to DEB-TACE was defined as no residual (TPost-RE ≤ 1) versus residual tumor enhancement (TPost-RE > 1).CTHA-EM analysis was conducted by a computer scientist (G.C.) and an imaging physicist (E.K.), independent of any interventional radiologists. Furthermore, such analysis was subsequently verified by an interventional radiologist board-certified in diagnostic and interventional radiology (B.C.O.) prior to the first follow-up imaging. Quantitative assessment was made by measuring the mean HU values in regions of interest (ROIs) on CTHA-EM images Fig. . ROIs weCTHA-EM-imaging-based response prediction was interpreted and recorded blinded to the first follow-up imaging (FUI). Finally, for tumors with residual enhancement on posttreatment CTHA-EM images, maximum intensity projection (5−10 mm thin) images were used to identify the potential feeding arteries supplying the residual tumor enhancement areas, which was defined by the presence of an artery leading to the area of residual tumor enhancement. In addition, pre-treatment CTHA imaging was evaluated to identify if such feeding arteries were present or if they were new collaterals that developed following DEB-TACE delivery.The first follow-up imaging after DEB-TACE was performed either with magnetic resonance imaging (MRI) or CT quadriphasic protocol according to the American Association for the Study of Liver Diseases guidelines . Two intp lower than 0.05 were considered statistically significant. Statistical analysis was performed by using commercially available statistical software .The study primary outcome measure was to evaluate the ability of CTHA-EM in predicting the per-tumor treatment response at the first follow-up imaging. Secondary outcome was to correlate the exact anatomical areas of residual tumor on CTHA-EM with the first follow-up imaging and to explore the capability of CTHA-EM in identifying the feeding arteries to residual tumors. Quantitative evaluation of tumor relative enhancement pre- and post-DEB-TACE (HU and percentage change) between treatment response cohorts was performed using the Wilcoxon rank-sum test. To calculate specificities, sensitivities, positive predictive values (PPV), and negative predictive values (NPV) with 95% confidence intervals (CI), we used cross-tabulation. Values of A total of 29 patients  with 46 HCCs  submitted to twenty-nine DEB-TACE sessions met the inclusion criteria. Demographic and clinical characteristics of all the patients are shown in Table TPre-RE and TPost-RE) showed significant differences between residual and no-residual enhancement groups after DEB-TACE . No significant differences in HU were observed in HCCs between no-residual and residual enhancement before treatment , indicating that the posttreatment CT attenuation values of the residual enhancement were similar to the pre-treatment values. Per-tumor response analysis based on CTHA-EM (TPost-RE) showed 18/46 (39.1%) HCCs that had residual enhancement, while 28/46 (60.9%) had no residual arterial enhancement.Quantitative evaluation of tumor relative enhancement . The first follow-up imaging showed complete response in 27 (58.7%), partial response in 17 (37.0%), and stable disease in 2 4.3%) of the 46 HCCs. Table .3% of thAmong the 18 HCCs with residual arterial enhancement at CTHA-EM, a total of 22 feeding arteries were identified on the posttreatment CTHA-EM images. These feeding arteries were not depicted on posttreatment two-dimensional DSA and were obscured by the presence of contrast agent stasis and the enhancement of the liver parenchyma on posttreatment CTHA Fig. . Of thesIn this proof-of-concept study, the proposed CTHA-EM method demonstrated a high accuracy in predicting HCC treatment response to DEB-TACE on the initial follow-up imaging. Moreover, among HCCs where complete response was not achieved, CTHA-EM was able to depict the feeding arteries supplying the residual tumor enhancement area. The deformable registration and subtraction of unenhanced and contrast-enhanced CTHA images allowed to differentiate true residual tumor enhancement from contrast agent stasis or pooling within the treated HCCs. Additionally, tumor relative enhancement metric provided an objective and reproducible method to determine residual tumor enhancement following DEB-TACE.Objective response  according to mRECIST is a relevant prognostic factor of survival in HCC patients undergoing TACE –32. Furti.e., need for further embolization), potentially improving the overall complete response rates following DEB-TACE.CTHA-EM can be a valuable intraprocedural tool to improve the treatment response prediction during HCC treatment with DEB-TACE. The challenges in predicting intra-procedural response to DEB-TACE are well illustrated in our present study, as 39.1% (18/46) of the treated HCCs had residual tumor on the first follow-up imaging, despite being deemed to have complete response at the DEB-TACE procedure’s completion per the interventional radiologist’s judgment based on the DSA and CTHA  images. This suggests that standard DSA and CTHA have a low negative predictive value in predicting incomplete DEB-TACE. Also, there is indication that CTHA-EM can provide valuable information on the identification of the residual tumor feeding artery, which could improve the treatment strategy (We believe that the high accuracy of CTHA-EM in identifying residual tumor demonstrated in the present study is related to several factors. First, the use of CT has many advantages as it provides reliable and reproducible imaging, allows standardized quantitative arterial enhancement assessment via HU quantification, offers easy correlation with the follow-up imaging, and facilitates better image quality with comparable or often lower radiation exposure compared to CBCT –40. SecoThis study has several limitations. First, the small number of cases reported might limit the generalizability of the present findings. The use of CT during hepatic arteriography for DEB-TACE has been part of our institution’s practice since 2016. The requirement of pre- and posttreatment CTHA images limited the case number for this proof-of-concept study. Such repeated CTHA imaging is not performed routinely in all cases, and it would translate into an increase on overall patient exposure to radiation. Therefore, we included patients from three different institutions from 2017 to 2020 to expand the number of cases to achieve sufficient power. Likewise, there is an inherent sampling bias as only patients undergoing first session DEB-TACE and who did not have extrahepatic arterial supply were included in this study. Third, correlating the results of CTHA-EM to treatment response on first follow-up imaging has its limitations, as tumor progression or response may occur beyond the first follow-up imaging. Fourth, the applicability of this method is limited to procedure rooms equipped with hybrid angio/CT system. Although the install base of angio/CT equipment in interventional radiology practice has seen a recent uptick, its current availability is widely limited to major academic centers, thus presently resulting in limited availability of the proposed method when implemented for intra-procedural use.In conclusion, the proposed CTHA-EM method can accurately and quantitively predict intraprocedural embolization endpoint and immediate treatment response after DEB-TACE on first imaging follow-up. In addition, it allows detection of the feeding arteries to residual tumor enhancement areas. The role of this method to personalize post-DEB-TACE imaging follow-up and its impact on tumor progression or response should be prospectively evaluated.Additional file 1: Supplementary Table 1. CTHA acquisition and reconstruction parameters.


Near-infrared photoimmunotherapy (NIR-PIT) is a newly developed cancer treatment modality based on a target-specific photosensitizer conjugate (TSPC) composed of an NIR phthalocyanine photosensitizer and an antigen-specific recognition system. NIR-PIT has predominantly been used for targeted therapy of tumors via local irradiation with NIR light, following binding of TSPC to antigen-expressing cells. Physical stress-induced membrane damage is thought to be a major mechanism underlying NIR-PIT-triggered photokilling. Notably, NIR-PIT can rapidly induce immunogenic cell death and activate the adaptive immune response, thereby enabling its combination with immune checkpoint inhibitors. Furthermore, NIR-PIT-triggered “super-enhanced permeability and retention” effects can enhance drug delivery into tumors. Supported by its potential efficacy and safety, NIR-PIT is a rapidly developing therapeutic option for various cancers. Hence, this review seeks to provide an update on the (i) broad range of target molecules suitable for NIR-PIT, (ii) various types of receptor-selective ligands for designing the TSPC “magic bullet,” (iii) NIR light parameters, and (iv) strategies for enhancing the efficacy of NIR-PIT. Moreover, we review the potential application of NIR-PIT, including the specific design and efficacy in 19 different cancer types, and its clinical studies. Finally, we summarize possible NIR-PIT applications in noncancerous conditions, including infection, pain, itching, metabolic disease, autoimmune disease, and tissue engineering. Owing to its high morbidity and mortality, cancer remains a great threat to human health despite the enormous amount of associated research. Photodynamic therapy (PDT) is a promising treatment modality as it offers efficient antitumor destruction with limited adverse effects via localized activation of a photosensitizer (PS) by light irradiation. PDT depletes cancer cells through reactive oxygen species generation or physical stress, consequently inducing cellular apoptosis or necrosis in vivo administration, the target-specific photosensitizer conjugate (TSPC) distributes into the tumor and binds to the antigen-expressing cell membranes . The activated T cells then elicit a host immune response against residual tumor cells following direct killing by NIR-PIT. This is the process of NIR-PIT-induced ICD A newly developed molecular-targeted antitumor phototherapy approach, called near-infrared photoimmunotherapy (NIR-PIT), has shown great potential in overcoming the challenges associated with PDT. NIR-PIT employs a tumor-specific binding ligand, such as a monoclonal antibody (mAb), mAb fragment, or small-molecule ligand that is conjugated to IRdye 700DX (IR700)—a silicon phthalocyanine dye—which is activated by NIR light to cause immunogenic cell death (ICD) s Figure A-B. Folls Figure C-D. The PS is a critical component of the “magic bullet” TSPC for NIR-triggered cytotoxicity. Among several PSs that have been investigated, the new-generation dye IR700 Figure B has manAn effect called “super-enhanced permeability and retention” (SUPR) was observed after NIR-PIT. As NIR-PIT rapidly kills perivascular cancer cells, tumor vascular permeability greatly increases, leading to much higher up to 24-fold) nanoparticle accumulation within the treated tumor 4-fold naThe ability of NIR-PIT to induce SUPR effects and trigger anticancer host immune responses has previously been reviewed. Its application in the local control of solid cancers, as well as certain molecular targets, antitumor mechanisms, and possible challenges in its implementation, have recently been discussed The accurate selection of molecules to be targeted by ligands is critical for cancer therapy. For most cancer-targeted therapies, significantly higher expression of the target molecules on cancer cells relative to that on normal cells is crucial for reducing unwanted off-target effects. However, in the case of NIR-PIT, researchers have primarily considered the distribution and function of the targeted antigens as NIR irradiation can be spatially confined to the tumor, and normal tissue is generally not damaged when using TSPC-delivered PS. Therefore, NIR-PIT has a wider target pool than other methods using other payloads that do not require external activation.. developed a TSPC by employing the anti-epidermal growth factor receptor (EGFR) antibody panitumumab (Pan) plus the anti-CD25 antibody basiliximab (Bas) for targeted delivery of IR700 to the ATAC4 cell line, with high expression of EGFR and low expression of CD25. No significant difference was observed in the therapeutic response to NIR-PIT using Pan-IR700 or the Pan-IR700/Bas-IR700 mixture in vitro. However, the cocktail of Pan-IR700 + Bas-IR700 resulted in greater inhibition of tumor growth and longer survival in tumor-bearing mice than either Pan-IR700 or Bas-IR700 alone. Analysis of the TSPC microdistribution in the tumors showed that Pan-IR700 was more peripherally localized despite the high expression of EGFR on tumor cells, whereas Bas-IR700, targeting the low-expression CD25, was distributed uniformly throughout the tumor, including the center where Pan-IR700 was not found Although the expression level of target molecules typically does not cause safety concerns in NIR-PIT, it does impact the therapeutic effect. That is, a higher expression level of the target will induce a more potent antitumor effect as more TSPCs can bind to the target and accumulate in the tumor site. However, using a high-affinity ligand/antibody recognizing a highly-expressed target molecule may not always prove beneficial. Deep penetration into the tumor parenchyma may be hampered by accumulation of high-affinity binding ligands/antibodies in the immediate proximity of blood vessels, a phenomenon known as the “binding site barrier” Figure  26. TherDespite a wide range of options, due to their overexpression and wide distribution in multiple cancers, EGFR and human epidermal growth factor receptor type 2 (HER2) are currently the most frequently used targets in NIR-PIT NIR-PIT can be designed to damage components of the TME, including vascular cells, cancer-associated fibroblasts (CAFs), and tumor-infiltrating immune cells, such as regulatory T cells (Tregs) and unwanted phagocytes + T cells and NK cells, which are present in the TME but suppressed by Tregs. Hence, by selective depletion of Tregs, CD25-targeted NIR-PIT not only induces regression of irradiated tumors, but also elicits a therapeutic response against distant non-treated tumors in a tumor-antigen-specific manner. Additionally, repeated applications of NIR-PIT are feasible, which may not be the case for ionizing radiation. Despite CD25 upregulation in activated CD8+ T and NK cells, CD25 expression on these effector cells is downregulated after repeated NIR-PIT following a 4-day interval + T cells, NK cells and DCs. Thus, by locally depleting multiple immunosuppressor cell types and significantly increasing CD8+ effector T cells in tumors, the CD73-targeted NIR-PIT + anti-PD1 antibody combination treatment could overcome acquired resistance to PD-1 blockade, and eradicate advanced tumor xenografts in mice, irrespective of CD73 expression levels in tumor cells Unlike tumor/vasculature-specific antibody-mediated NIR-PIT, CD25-targeted NIR-PIT has been used to selectively eradicate tumor-infiltrating Tregs—a type of immunosuppressive cell with high CD25 expression—to treat a broad range of cancers in murine models, including lung, colon, and prostate cancers TSPCs recognizing different antigens can be combined in NIR-PIT treatment to enhance the therapeutic effects TSPCs with different sizes, affinities, and pharmacokinetics can influence the therapeutic effects of NIR-PIT. An intact antibody has a relatively large size, and thus exhibits poor penetration in solid tumors, limiting the therapeutic efficacy. In this regard, genetically engineered small antibody fragments may retain a high affinity for the antigen while improving the intratumoral delivery of TSPCs. Moreover, the smaller size can enable rapid accumulation of TSPC in the tumor along with rapid systemic clearance. Generation of TSPCs comprising with a diabody (Db), minibody (Mb) Shirasu et al. reported an interesting universal TSPC construction, expanding the applicability of NIR-PIT. They conjugated IR700 with NeutrAvidin, which specifically binds to a range of biotinylated tumor-homing molecules. This TSPC indirectly binds to targeted cells with the help of anti-CEA or anti-EpCAM biotinylated antibodies and exhibits cytotoxicity levels, after light irradiation, similar to those elicited by other NIR-PITs, which TSPCs bind directly to the cell membrane As discussed previously, the affinity of TSPC can affect the antitumor response of NIR-PIT due to the “binding site barrier” in vitro. However, Pan-IR700 exhibited an antitumor response superior to that of cet-IR700 in vivo. Examination of the biodistribution revealed that cet-IR700 had lower tumor accumulation and more rapid hepatic catabolism than Pan-IR700 The pharmacokinetics of TSPC also affect NIR-PIT efficacy. A previous study compared TSPCs constructed using different anti-EGFR antibodies. Those TSPCs with two antibodies, namely, cetuximab (cet) and panitumumab (Pan), exhibited equal affinity and identical phototoxicity 2-guided TSPC. F(ab')2-mediated NIR-PIT recruits a higher number of infiltrating lymphocytes to the tumor. The slower clearance of anti-CD25-IgG-IR700 might account for its lower efficacy, as the sustained circulation of anti-CD25-IgG-IR700 can impair the binding of activated effector T cells to IL-2, a cytokine that promotes T cell proliferation Notably, when TSPCs specifically bind to their targeted receptors, they may transiently block receptor function, thereby, altering therapeutic efficacy. In a study that eradicated Tregs using anti-CD25-targeted NIR-PIT, an IgG-directed TSPC was reported as less effective than an F(ab')22) of the light source is not as critical to the effectiveness of NIR-PIT as the total light dose (J/cm2) NIR light is an important component of NIR-PIT. While the light dose in NIR-PIT is typically between 50 and 150 J/cmResearchers are currently seeking improved strategies to deliver NIR light. In addition to delivering NIR irradiation from outside the body, endoscopic delivery is also being explored in vitro and antitumor effects against large tumor xenografts (~200 mm3) in vivo.A commonly used strategy to improve the antitumor effect is combining NIR-PIT with other therapeutic agents, such as nanodrugs In this section, we review the application of NIR-PIT in cancer treatment Figure -3. The tIn vitro studies suggest that aggressive BL is more sensitive to NIR-PIT than indolent BL. In animal models using two human aggressive BL cell lines (Daudi and Ramos), NIR-PIT significantly inhibits tumor growth and prolongs survival compared with control groups CD20 is a B-cell specific antigen highly expressed in many B cell lymphomas (BLs) and was, therefore, selected as an important target for treating BL EGFR is highly expressed in 74% of bladder cancer tissue specimens Researchers have also made efforts to develop a larger animal model for translational studies and to investigate cystoscopy for NIR light delivery. As spontaneous bladder cancer in dogs closely mimics human invasive bladder cancer in vitro and in vivo. However, 4/11 mice died within 48 h following irradiation with the higher dose light (100 J/cm2) in the combination group. Indeed, excessive light exposure or TSPC dosage beyond the maximum tolerated doses (MTDs) can result in death of mice. Thus, the potential treatment toxicity with drug dose-escalation must be considered to achieve the optimal antitumor response. Subsequent toxicity assays indicate that tumor lysis syndrome (TLS), caused by massive tumor necrosis, provoked a strong systemic inflammatory response in the mice that died HER2 is a member of the tyrosine kinase EGFR family that promotes cell proliferation, differentiation, and motility. It is commonly overexpressed on various types of cancer cells, and has been identified in 40% of bladder cancer specimens CD47—a ligand that transmits inhibitory signals for macrophage phagocytosis—is overexpressed on bladder cancer cells, however, is absent in the normal luminal urothelium ex vivo experiments showed that although the transmittance of NIR light was only 4.52%, this therapy could potently destroy cancer cells behind the rib Nakamura et al. chose an explanted bovine rib as a model to determine whether NIR-PIT can effectively kill cancer cells within bones. Their EGFR:03115-IR700DX). This conjugate showed high specificity for binding to EGFR and significant therapeutic efficacy after light irradiation, both in vitro and in a xenograft model of GBM Considering that EGFR is overexpressed in approximately 50% of primary glioblastomas (GBMs), it represents an interesting target for GBM therapy in vitro and in a xenograft model G protein-coupled receptors (GPCRs), including US28 and type 2 cannabinoid receptor (CB2R), are abnormally overexpressed in multiple tumors, including brain tumors CSCs are undifferentiated tumor cells with high self-renewal capacity, limited differentiation capability, and high motility HER2 is highly expressed in approximately 20% of human breast cancers and is generally associated with poor outcomes in vivo after irradiation at a high light dose (>100 J/cm2) Anti-EGFR-based NIR-PIT has also been investigated for the treatment of breast cancer. In an orthotopic breast tumor model, panitumumab conjugated with IR700 for NIR-PIT immediately induced the killing of >90% cells TNBC is defined by the lack of expression of estrogen receptor (ER), progesterone receptor (PR), and HER2, and is associated with shorter relapse-free intervals and lower overall survival than other subtypes. Moreover, no targeted therapy is currently available for TNBC, in contrast to ER-positive or HER2-positive breast tumors. Fortunately, 50-70% of TNBCs express EGFR in vitroA cocktail of NIR-PIT targeting three different receptors has also been investigated for the treatment of TNBC. These receptors, including EGFR, EpCAM, and chondroitin sulfate proteoglycan 4 (CSPG4), are abundantly expressed in TNBC, but only at low levels in normal tissues The high recurrence and metastasis rates of TNBC are due in part to the presence of CSCs, which are resistant to conventional therapy Type I transmembrane protein tumor-associated calcium signal transducer 2 (TROP2) has been tested as a therapeutic target in pancreatic carcinoma (PC) and CCA. NIR-PIT with an anti‐TROP2 antibody significantly inhibits PC and CC xenograft tumor growth. In the same study, TROP2 was identified in 40% of PC samples and 46% of CCA samples, indicating that half of the patients could be covered Elekonawo et al. evaluated the effects of CEA-targeted NIR-PIT in nine CRC cell lines expressing different levels of CEA. Their results indicate that the antitumor efficacy of NIR-PIT is proportional to CEA expression level ) exhibits rapid and specific killing of GPA33-positive colorectal tumor cells after NIR laser light irradiation in vitro. In mice bearing GPA33-positive tumor xenografts, A33scFv-IR700-mediated NIR-PIT exhibits significant antitumor effects after a single NIR exposure Moreover, glycoprotein A33 antigen (GPA33), which is overexpressed in human CRC, has also been investigated as a target for NIR-PIT 3) with both chemotherapy MDR and TRAIL resistance Since EGFR is also highly expressed in most CRC cells, an EGFR-targeting affibody was utilized as an NIR-PIT agent combined with long-acting tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) to treat chemotherapy MDR and TRAIL resistance in CRC. In this study, EGFR-targeted NIR-PIT significantly increased the sensitivity of MDR cells to TRAIL, and the combination therapy efficiently eradicated large CRC xenografts .NIR-PIT is suitable for the treatment of skin disorders as the light can be directly irradiated. In the case of CSCC, EGFR in vitro. Researchers have also found that the therapeutic effects of NIR-PIT can be further enhanced using a TKI to upregulate the expression of growth factor receptors in vitro and in vivoGrowth factor receptors, including EGFR and HER2, can act as targets of NIR-PIT in EC. TSPCs targeting these growth receptors showed good phototoxicity in vivo can be attributed to the difference in cell killing mechanisms, namely, necrotic membrane damage and apoptotic cell death Several studies have tested NIR-PIT to treat peritoneal dissemination of gastric cancer because NIR light can be distributed to multiple cancer sites simultaneously in the peritoneal cavity via endoscopy. Given that approximately 20% of gastric cancers express HER2, these studies utilized anti-HER2 NIR-PIT in vitro and in mouse models Gastrointestinal stromal tumors (GISTs) are the most common type of submucosal tumors of the gastrointestinal tract, with up to 90% of GISTs overexpressing c-KIT  VEGFR-2 expressed on the vascular endothelium has also been evaluated as a potential target of NIR-PIT for gastric cancer. In fact, anti-VEGFR2-mediated NIR-PIT induces damage in the tumor neovasculature of xenograft tumors. Compared with anti-HER2 NIR-PIT, VEGFR2-targeted NIR-PIT has a stronger antitumor effect, although this approach leads to a lower amount of TSPC inside the tumor. However, vessel destruction may fail to induce SUPR effects and the subsequent immune response achieved with anti-HER2 NIR-PIT. Moreover, although VEGFR2 is also expressed in normal tissues, NIR light can be applied locally, thus limiting normal tissue toxicity in vitro, and in an in vivo model of residual disease after incomplete resection. Mice that received surgical treatment plus adjuvant NIR-PIT showed a significant (P < 0.001) reduction in tumor regrowth 30 days post-NIR-PIT in both the 50% and 90% subtotal resection groups. However, tumor recurrence ultimately occurred in all treated mice. Hence, a more potent NIR-PIT regimen, such as repeated light exposure, may be needed in vivoDue in part to the complex topography of the regional anatomy and the abundance of vital structures, the recurrence rate of squamous cell carcinoma of the head and neck (SCCHN) remains high even after administration of the gold standard treatment, i.e., surgical resection plus adjuvant radiotherapy, or chemoradiation et al. compared NIR-PIT using the whole IgG antibody anti-GPC3 YP7 with that using the human heavy-chain antibody anti-GPC3 HN3 and found that both groups showed significant inhibition of tumor growth in vivo. However, the HN3-IR700 conjugate provided a deeper, and more homogeneous, distribution in tumors thanYP7-IR700, without compromising the affinity of HN3 for GPC3. Moreover, HN3-IR700 retained its capacity to induce antibody-dependent cell-mediated cytotoxicity (ADCC). Hence, researchers may prefer to use HN3 rather than YP7 in NIR-PIT for HCC treatment. Considering that light delivery can be problematic in HCC, light probes placed through vessels or inserted directly through the skin into the liver  could be necessary for effective NIR-PIT Glypican-3 (GPC3) is an attractive antigen for treating HCC as its expression in HCC is higher than that in normal liver P < 0.01 at all time points) P = 0.002 vs. TSPC) and prolonged survival (P < 0.0001) in vitro and a remarkable decrease in tumor volume in a xenograft model The lungs are considered suitable for light therapy due to the relative ease of light penetration into deep air-pockets without significant light attenuation. In a transgenic model of spontaneous lung cancer, tumor growth was significantly inhibited in the anti-EGFR NIR-PIT group compared with the control group (P < 0.0001) without damaging non-targeted tumors or normal lung tissues 3 in volume Considering that the lungs are among the most common sites for developing metastases, NIR-PIT has also been used to treat lung metastases. In fact, in a murine model, anti-HER2 trastuzumab-based NIR-PIT demonstrated a significant reduction in early-stage lung metastatic disease , a type I transmembrane glycoprotein, is used as a specific pathological diagnostic marker for MPM. Anti-PDPN antibody NZ-1-mediated NIR-PIT induces light-dose-dependent cytotoxicity3), whereas no significant difference was detected in large xenografts (330.80 ± 36.12 mm3) Melanoma is one of the most fatal types of skin cancer. Given that CD146 is upregulated in melanomas and is associated with progression and metastasis + T cells, resulting in greater tumor growth inhibition with prolonged survival Furusawa et al. investigated the efficacy of CD29-targeted NIR-PIT in the highly pigmented melanoma model. CD29  is a cell surface glycoprotein that is expressed on both melanoma and cytotoxic T cells. CD29-targeted NIR-PIT efficiently suppresses tumor growth in the pigmented melanoma model. More importantly, NIR-PIT does not cause damage to immune cells at the treatment site while preferentially killing proliferative cancer cells. Moreover, the combination of an anti-CTLA4 antibody and CD29-targeted NIR-PIT increases the infiltration of CD8Virus-like particles (VLPs) and IR700 have been employed to construct a TSPC (AU-011) for the treatment of primary uveal melanoma, leading to significant dose-dependent tumor necrosis after NIR irradiation. According to Kines et al., VLPs were prepared based on the HPV capsid protein. VLP attachment and internalization by targeted cells is mediated by heparan sulfate proteoglycan (HSPG) expressed on multiple tumor cell types. VLPs have two advantages over antibodies for NIR-PIT treatment. First, they exhibit multivalent binding, which enables stronger interaction with target cells. Second, a large number of IR700 dye molecules can be readily conjugated to a single VLP, leading to a higher concentration of PS on the cell surface + cells in vitroMF is a low-grade T-cell lymphoma, accounting for approximately 53% of primary cutaneous lymphomas P = 0.0037) Patients with intraperitoneal tumor dissemination of ovarian carcinoma, commonly present at an advanced stage and have a poor prognosis Integrins are transmembrane cell adhesion proteins that can act as signaling receptor proteins to regulate cancer progression by binding to extracellular matrix (ECM) components. Additionally, some integrins are overexpressed in ovarian cancer, neovasculature, and on immunosuppressive lymphocytes P = 0.001) and 2/7 mice (P = 0.03), compared with 7/7 mice and 6/7 mice respectively, in the BLS alone group. Significant differences in the tumor growth rate, tumor size, and tumor weight between the two groups persisted even several weeks after surgery P = 0.05). Moreover, the average weight of recurrent tumors was significantly lower in BLS+PIT-treated mice than in the BLS alone group (P = 0.015) Pancreatic cancer cells express high levels of CEA Cadherin-17 (CDH17 or CA17), a calcium-dependent transmembrane glycoprotein, is highly expressed in many gastrointestinal cancers including pancreatic adenocarcinoma. CDH17-targeted NIR-PIT significantly inhibits tumor growth in a pancreatic adenocarcinoma xenograft model after three cycles of treatment Prostate-specific membrane antigen (PSMA) is an attractive target for the treatment of PCa using NIR-PIT as it is highly expressed in prostate cancer cells and in the neovasculature. PCa can be illuminated with NIR light either directly or via a transurethral catheter The potential efficacy and safety of NIR-PIT make it a promising modality for cancer therapy, leading to its evaluation in various clinical trials worldwide. More specifically, in the United States (US), a phase I/IIa clinical trial of NIR-PIT based on a conjugate comprising cetuximab and IR700  has recently concluded. Results indicate that a manageable safety profile and substantial antitumor responses were obtained in locoregional, recurrent HNSCC patients who had previously exhibited unsatisfactory therapeutic responses to traditional clinical therapy Staphylococcus aureus (MRSA) cells after light irradiation. With the help of the enhancing agent potassium iodide, the anti-IsaA NIR-PIT can eliminate a high MRSA titer, regardless of the strong antioxidant activity of plasma Candida albicans. In a cutaneous infected mouse ulcer model, TSPC led to skin recovery after NIR irradiation with minimal damage to the uninfected epithelium The increasing prevalence of antimicrobial-resistant bacteria necessitates the development of alternative antimicrobial strategies, including targeted antimicrobial photodynamic therapy (aPDT). Bispo et al. used an antibody against the immunodominant staphylococcal antigen A (IsaA) for IR700 conjugation, which successfully targets and destroys methicillin-resistant Innocuous touching provokes severe pain in patients with mechanical allodynia, a major symptom of neuropathic pain. Peripheral sensory neurons expressing receptor tyrosine kinase B (TrkB) can sense the lightest touch under basal conditions and have been identified as necessary for mechanical allodynia in mice after nerve injury. In models of neuropathic pain, brain-derived neurotrophic factor  selectively mediated NIR-PIT and effectively eliminated TrkB-positive sensory fibers, thus reversing mechanical allodynia in multiple types of neuropathic pain In summary, NIR-PIT can directly attack cells to bypass the complex signaling pathways underlying pain responses. Moreover, the TSPC dosage, light intensity, and frequency of application are adjustable, thus allowing for personalized therapeutic regimens. Indeed, the long-lasting efficacy of a single photoablation treatment regimen has the potential to reduce toxicity compared with other therapies.Chronic itch causes an urgent desire for scratching and subsequent skin damage, which is not only difficult to treat, but also impacts the quality of life to the same extent as pain Uncontrolled production of insulin by pancreatic β-cells can cause hyperinsulinemic hypoglycemia Fibroblast-like synoviocytes (SFs) are a unique cell type that distinguishes RA from other inflammatory diseases affecting the joints SSc, also called scleroderma, is a complex autoimmune disease characterized by inflammation, vasculopathy, and fibrosis of the skin and internal organs. This fibrosis is partially caused by the presence of activated myofibroblasts (myo) in the tissues  ex vivo, and from mixed populations in tumor models without damaging non-targeted cells It is desirable, yet difficult, to eliminate a specific type of cell from mixed cell cultures or tissues without damaging adjacent cells. By employing targeted antibodies and spatially selective irradiation, NIR-PIT could eliminate specific cells from mixed 2D/3D cell cultures or tissuesDespite the very promising therapeutic effects, a few potential limitations and challenges remain when considering NIR-PIT implementation. First, light exposure can cause potential toxicity to organs located in proximity to the tumor. In fact, mild peri-tumor dermal edema is one of the most common treatment emergent adverse events (TEAE) reported in patients treated with NIR-PIT. Meanwhile, low-grade, reversible skin photosensitivity was observed in a small proportion of treated patients in vitro and in vivo animal models, showing promising therapeutic effects and leading to its application in clinical trials. A wide range of targets can be selected for binding by NIR-PIT agents. Often, tumor-associated cell-surface antigens or glycoproteins, such as EGFR, HER2, or PSMA, are used as tumor-homing targets, given that they are overexpressed on many tumors. In the TME, targets expressed on the non-tumoral cells that support and maintain the tumor have also been investigated. Moreover, multiple antigens can be targeted simultaneously using a cocktail of TSPCs in an NIR-PIT setting.NIR-PIT has been widely studied in various types of cancer, and noncancerous diseases, both Antigen-specific recognition agents, including antibodies and their fragments, peptides, protein ligands, and small molecules have been employed in the construction of TSPCs. Each has different advantages and can be selected depending on the function and cellular distribution of the antigen.NIR light is also a critical factor in NIR-PIT. Light dose, power density, and source influence the therapeutic efficacy. Light delivery systems are of great importance for NIR-PIT. Although NIR light is most commonly delivered by an external light source, endoscopy and wireless implantable light systems have also been used. NIR-PIT, as an adjunctive therapy, could eliminate residual tumor cells after BLS, which is also feasible to reduce metastasis and local recurrence. NIR-PIT can be combined with other modalities to achieve improved antitumor effects. The advantages of these combinations can be ascribed to SUPR effects and other synergistic antitumor mechanisms, such as enhancing antitumor immunity by combination with an immune checkpoint blockade. Recent studies have reported only minimal adverse events associated with NIR-PIT. However, the safe range of TSPC doses and light doses should be further explored. Overall, NIR-PIT holds great promise for clinical applications to treat cancer and other non-malignant diseases with targetable antigens/receptors. However, the toxicity and safety of NIR-PIT must be fully investigated before it can be translated into clinical settings.


Ultrafast atomic vibrations mediate heat transport, serve as fingerprints for chemical bonds and drive phase transitions in condensed matter systems. Light pulses shorter than the atomic oscillation period can not only probe, but even stimulate and control collective excitations. In general, such interactions are performed with free-propagating pulses. Here, we demonstrate intra-cavity excitation and time-domain sampling of coherent optical phonons inside an active laser oscillator. Employing real-time spectral interferometry, we reveal that Terahertz beats of Raman-active optical phonons are the origin of soliton bound-states – also termed “Soliton molecules” – and we resolve a coherent coupling mechanism of phonon and intra-cavity soliton motion. Concurring electronic and nuclear refractive nonlinearities generate distinct soliton trajectories and, effectively, enhance the time-domain Raman signal. We utilize the intrinsic soliton motion to automatically perform highspeed Raman spectroscopy of the intra-cavity crystal. Our results pinpoint the impact of Raman-induced soliton interactions in crystalline laser media and microresonators, and offer unique perspectives toward ultrafast nonlinear phononics by exploiting the coupling of atomic motion and solitons inside a cavity. Here, the authors demonstrate intra-cavity excitation and time-domain sampling of coherent optical phonons inside an active laser oscillator. They discover that Terahertz crystal vibrations link successive ultrashort solitons which offers an approach to highspeed Raman spectroscopy inside laser cavities. The probing of atomic motion on its natural timescale unveils phonon lifetimes, vibrational dephasing and couplings in condensed matter3. Particularly in transparent media, ultrashort pump pulses can impulsively stimulate coherent phonon motion via Raman scattering non-resonant with the electronic system. Impulsive stimulated Raman excitation allows for preparing, manipulating and controlling coherent phonon motion6—and may occur at every roundtrip inside an active ultrafast laser cavity, as we demonstrate in the following. Atomic lattice dynamics commonly remain hidden due to the fast phonon decay compared to the cavity roundtrip period of several nanoseconds. Here, we study the inter-soliton motion on femto- and picosecond timescales during laser operation based on two circulating solitons and unravel the link between impulsive stimulated Raman excitation and the largely unresolved interaction of multiple ultrashort pulses. In particular, we identify Raman-active optical phonons as the key drivers of soliton binding9 and complex inter-soliton trajectories10 inside a laser cavity, as sketched in Fig. 13.Time-resolved sampling of coherent excitations comprises a key concept of pump-probe spectroscopy in experimental quantum physics and ultrafast sciences15, the scheme has been applied to nonlinear behavior inside ultrafast laser oscillators, opening up views into the buildup of soliton mode-locking and complex multi-soliton interactions in real-time18. Moreover, temporal solitons are observed to form stable and meta-stable bound states—termed “soliton molecules”—with non-commensurate temporal separation distances, spanning from picoseconds down to few 10-femtoseconds. Deterministic switching between such soliton molecules upon external control has also been demonstrated19. The temporal soliton separations in these systems match various earlier reports on double-pulsing in Kerr-lens modelocked Ti:sapphire lasers—the underlying mechanisms, however, have remained elusive9.Recently, real-time spectroscopy based on the time-stretch dispersive Fourier transform (TS-DFT) has enabled high throughput measurements in laser science and nonlinear optics, yielding consecutive single-shot spectra exceeding 100 million frames per second. Following observations in passive nonlinear systems10. According to the common design of mode-locked Titanium-doped sapphire oscillators, the crystal serves as a broadband gain medium and as an ultrafast nonlinear amplitude modulator, enabling passive mode-locking via intensity-dependent focusing due to the spatial Kerr-lens effect21. Upon detuning of the resonator stability from single-pulse operation, mode-locking of two solitary pulses is readily obtained, which circulate principally independently inside the resonator. Yet, interactions based on the laser gain saturation and nonlinear refraction induce different group velocities and, thus, generate relative motion of both pulses22. As a result, the two solitons autonomously scan through their temporal separation and effectively perform degenerate pump-probe sampling in an all-optical and autonomous fashion. For large temporal separations of several hundred picoseconds, such relative motion is perceivable via fast real-time photodetection of the output intensity over many roundtrips.In this work, we analyze real-time measurements of transient multi-soliton dynamics in Kerr-lens modelocked lasers, based on experimental data and previous reports10 At each roundtrip, we map the Fourier amplitudes of the respective spectral interferograms that represent field-autocorrelations. Thus, the side peak of the symmetric dataset reveals the relative soliton separation (mirror-symmetric negative delays omitted). We observe that both solitons approach via steps at separations in the picosecond to 100 fs range. Such meta-stable separations appear incommensurably spaced and exhibit varying degrees of stability. In order to analyze these dynamics, we generate a histogram of dynamic separations by integrating the roundtrip-resolved autocorrelation peaks along consecutive traces on the real-time axis. The result is shown in Fig. The critical dynamics on pico- to femtosecond separations are not resolved via direct photodetection, but can be tracked on a continuous, single-shot basis employing TS-DFT and spectral interference. We track the inter-soliton motion via recording single-shot spectra that encode soliton separations and relative phases via spectral interference, as illustrated in Fig. 2O3) and corresponds to the crystal structure of the rhombohedral space group R-3c. The birefringent crystal is cut at Brewster’s angle and is aligned with the optical axis parallel to the linear polarization of the beam inside the crystal. Referring to Porto’s notation, the Raman selection rules for the corresponding X(ZZ)X-geometry allow for accessing two A1g modes given by optical Terahertz phonons at frequencies of 645 1/cm and 418 1/cm or 16 THz and 13 THz, respectively24. Following the impulsive Raman excitation by the first soliton at delay Given these observations, we can directly unravel the underlying origin of the observed soliton molecule motion by relating the data to the temporal Raman response of the laser host medium, shown in Fig. ϕ(τ) between two pulses with fields Moreover, we now demonstrate the time-domain Raman spectroscopy of phonon vibrations based on this single trajectory of two approaching solitons, captured within an acquisition time below 170 µs. The scheme is enabled by introducing “consecutive double-referencing” for sensing delay-dependent relative phase changes: Whereas the Fourier amplitudes of the spectral interferograms yield the field-autocorrelations, the fringe positions encode the relative phase ∆n, it yields the relative phase 23 (orange). The time-domain data yield complex-valued Raman spectra via Fourier transformation25 and the resultant Raman spectrum is presented in Fig. 26 . The quality of the Raman signal depends on cavity alignment, as the intrinsically nonlinear laser behavior allows for diverse sets of trajectories at different operation parameters. Yet, characteristic Raman signatures are found in every dual-soliton trajectory, whereas laser instabilities and noise may induce deviations of sampled Raman waveforms  measurements, we obtain an upper limit of the intrinsic nuclear versus electronic contribution to 30, the measured intra-cavity Raman phase oscillation of ~0.05 rad yields signal enhancements exceeding a factor of five , respectively. Spectra are dispersed using 600 m length of single mode optical fiber, enabling the resolution of soliton separations up to 2000 fs. Extra-cavity reference measurements are performed via SRTBC and Kerr-lens sampling with lockin-detection. Additional data, details on data analysis and simulation are provided in the Supplementary Information