Senescence Programs Shape the Chronic Wound Microenvironment.

1. Co-chaired this panel2. University of South Florida, Tampa, FL3. Healthpoint Ltd., Fort Worth, TX4. University of California, San Francisco, CA5. University of Texas Medical Branch, Galveston, TX6. University of Cardiff, Cardiff, Wales, UK7. University of Pennsylvania, Philadelphia, PA8. Private practice, Warren, PA9. Private practice, Tamarac, FL10. University of Pittsburgh, Pittsburgh, PA11. St. Louis University, St. Louis, MO, and12. Washington University, St. Louis, MO

Senescence, largely initiated by unrepaired DNA damage, is initially sustained by upregulated p53. Although senescent cells enter proliferative arrest, early p53 supression may result in senescent cells re-entering the cell cycle. Senescent cells exhibit a senescence-associated secretory phenotype (SASP) that impacts the cell microenvironment, often promoting cell transformation. SASP molecular composition may be cell-specific and dependant on the type of SASP-producing cell. As growth hormone (GH) is induced by DNA damage, we elucidated whether GH is induced in senescent cells. Non-pituitary GH (npGH) synthesized locally in peripheral tissues is identical to endocrine GH1 produced by the pituitary and acts through autocrine/paracrine mechanisms via the widely expressed GH receptor that recognizes both pituitary GH and npGH ligands. We show that npGH is induced in aging human colon tissue, and in human non-tumorous colon cells and in human 3-dimensional intestinal organoids in response to oncogene-, therapy-, and/or replicative-induced senescence. Furthermore,DNA-damage-induced npGH is secreted from senescent cells, constituting a SASP component. In senescent cells, DNA damage is not repaired with fidelity, and we show that induced npGH suppresses DNA damage responses by attenuating phosphorylation of ATM, DNA-PKc, p53 and Chk2, resulting in p53 suppression and accumulation of damged DNA. Autocrine npGH also triggers senescent cell proliferation with increased Ki67 and BrdU incorporation. As proliferating cells with accumulated unrepaired DNA damage may acquire oncogenic mutations,we assessed npGH actions on cell transformation. We show that senescent colon cells expressing npGH form colonies in soft agar,while GH depletion by shRNA downregulates Ki67 and decreases colony formation and size, suggesting that npGH enables senescent cell transformation. Consistent with a SASP function, induced paracrine npGH also suppresses the p53/p21 pathway, triggering proliferation and exacerbates DNA damage in neighboring non-senescent cells. To further explore mechanisms underlying npGH induction we tested the role of the SASP chemokine CXCL1 which attracts immune effectors to eliminate senescent cells. CXCl1 is shown to induce npGH in senescent hNCC and in intestinal organoids, while GH, in turn, suppresses CXCL1, likely by inhibiting NFκB, a CXCL1 transcription factor. Both colon CXCL1 and NFκB are more abundant in GH-receptor knockout mice devoid of GH signalling, while mice bearing GH-secreting xenografts exhibit decreased colon CXCL1 abundance. Conclusions: The results elucidate a heretofore unappreciated GH action, whereby npGH, as a SASP component, attenuates senescent cell elimination by inhibiting CXCL1, and contributes to a tissue microenvironment favoring age-associated DNA damage accumulation and epithelial cell transformation. Citation Format: Vera Chesnokova, Svetlana M. Zonis, Robert Barrett, Shlomo Melmed. Growth hormone as a SASP component [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5683.

Senescence represents a fundamental response to cancer therapy. Accumulating senescent cells contribute to the deleterious outcomes of cancer therapy including cancer relapse, effects that may be largely mediated by the Senescence-Associated Secretory Phenotype (SASP). In this work, we show that tumor cells induced into senescence by etoposide retain proliferative capacity based on their capacity to generate proliferating colonies in culture as well as giving rise to viable tumors in vivo. Using a flow cytometry-based enrichment approach based on enlarged size and expression of Senescence-Associated β-galactosidase (SA-β-gal), we were able to utilize real time imaging to establish the re-emergence of non-small cell lung cancer cells from senescence-based arrest and the generation or proliferating daughter cells (i.e. self-renewal). Moreover, we implemented High-Speed Live-Cell Interferometry (HSLCI) to provide a single-cell lineage tracking of dividing senescent cells. The recovery from senescence was accompanied by resolution of several senescence-associated hallmarks, specifically SA-β-gal activity, p21Waf1/Cip1 and several components of the SASP (IL-1β, IL-6 and CXCL1). Our data suggests that Therapy-Induced Senescence (TIS) may ultimately be a transient process in that at least a subpopulation of tumor cells can recover proliferative capacity. We further demonstrate that the senolytic agent, ABT263, which has been shown to eliminate senescent cells from aging-related animal models can also eliminate senescent tumor cells that persistent after exposure to chemotherapy by shifting the response towards apoptotic cell death. Furthermore, sequential administration of ABT263 interferes with the ability of tumor cells induced into senescence by chemotherapy to recover growth potential. These studies suggest that senescent tumor cells can potentially contribute to cancer relapse by acquiring proliferative properties. The use of senolytic agents after induction of senescence by conventional or targeted therapies allows for the clearance of residual (possibly dormant) senescent tumor cells, which could serve to suppress disease recurrence and cancer mortality. Citation Format: Tareq Saleh, Liliya Tyutyunyk-Massey, Graeme F. Murray, Moureq R. Alotaibi, Ajinkya S. Kawale, Zeinab Elsayed, Scott C. Henderson, Vasily Yakovlev, Lynne W. Elmore, Amir Toor, Hisashi Harada, Jason Reed, Joseph W. Landry, David A. Gewirtz. Elimination of senescent tumor cells by ABT263 interferes with proliferative recovery and provides a two-hit therapeutic approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 901.

Cellular senescence is characterized by stable cell cycle arrest and a secretory program that modulates the tissue microenvironment. The aberrant accumulation of senescent cells in aged and cancerous tissue triggers inflammatory signaling through a senescence-associated secretory phenotype (SASP), promoting aging and tumor progression. Pharmacologically, clearing senescent cells has been shown to have promising effects against cancer and age-related pathologies in preclinical models as well as human pilot clinical trials. However, current senescent cell elimination strategies are focused on the rare senescence targets that are under-represented on normal cells. To date, there are no universal senescence cell specific markers. Therefore, the use of senolytics in the clinic is still limited due to their cytotoxicity to normal cells. A more effective senescence cell targeted approach is needed to reduce side effects and increase the efficiency of senescence cell removal. Conditionally Active Biologics (CAB) technology is a proprietary platform that is unique in its ability to selectively activate in the context of diseased tissues, but not normal tissues, taking advantage of the acidic pH conditions in the inflammatory tumor/aged tissue microenvironment. Senescent cells, like tumor cells, are dependent on glycolysis for energy metabolism, and thus generate a glycolytic microenvironment (GME) that resembles that of the tumor microenvironment. Since our CAB technology is already being deployed in cancer therapy by targeting cell surface markers such as AXL, ROR2, and CTLA4 in the clinical stage, we explored whether CAB technology will allow selective removal of senescent cells in SASP-associated microenvironments such as the GME. To identify senescence-specific surface antigens that are broadly and specifically upregulated in senescent cells, we screened our CAB cell surface antigen library in different senescence models including CDK4/6 inhibitor-induced senescent human breast cancer cells, human lung fibroblast cells, as well as in vivo aging mouse tissues and chronic kidney disease mouse model. We have identified several novel senescence markers which show significantly elevated expression in senescent cells. Furthermore, in vitro binding assays demonstrated that CAB antibodies targeting these senescence markers have no or very low binding to the target antigen on senescence cells in alkaline physiological conditions but have strong binding in glycolytic/acidic SASP conditions. Moreover, we performed antibody-dependent cell-mediated cytotoxicity assays for evaluating the functional activities of CAB antibodies against the senescence targets. Our data demonstrated that CAB antibodies were more potent against senescent cells compared to proliferating cells and displayed high selectivity for the glycolytic/acidic microenvironment condition. In conclusion, CAB technology should provide a new generation of biologics with an increased safety margin and therapeutic index in the clinic targeting senescence cells in cancer and age-related diseases. Citation Format: Jian Chen, Jing Wang, Matt Lucas, Haizhen Liu, Christina Wheeler, Kyrie Johnson, Kathryn Woodard, Cathy Chang, Gerhard Frey, William J. Boyle, Jay M. Short. Targeting senescence cells in cancer and aging by conditionally active biologic therapeutics [abstract]. In: Proceedings of the AACR Special Conference: Aging and Cancer; 2022 Nov 17-20; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_1):Abstract nr B022.

Cellular senescence is a state of stable growth arrest in response to stress, which is a fundamental process of biological aging. They secrete products, the Senescence-Associated Secretory Phenotype (SASP), which consists of inflammatory cytokines, chemokines, growth factors and matrix remodeling proteins. Senescent cells accumulate with advancing age and partial elimination of senescent cells can reverse age-related dysfunction and increase mean lifespan in mice. However, it is not clear whether components of the SASP can be measured in human plasma and serve as aging biomarkers. Here we generated a candidate panel of senescence markers based on a multiplexed bead-based assay of proteins secreted by senescent preadipocytes, endothelial cells, fibroblasts, myoblasts, preadipocytes, and epithelial cells compared to non-senescent controls. The SASP undoubtedly varies by cell type; however, we observed that multiple components of the SASP are conserved. We then assessed circulating SASP components in human plasma samples from Mayo Clinic Biobank participants (n=280, 20 male and 20 female per decade, age 20-90) using the same method. We confirmed that components of the SASP can be quantified in human plasma with the multiplexed bead-based assay and observed several SASP components robustly increase with chronological age in humans. Our study illustrates that senescence-associated secretome components are detectable in human plasma and could potentially serve as biomarkers of systemic aging and senescent cell burden.

Oridonin inhibits SASP by blocking p38 and NF-κB pathways in senescent cells

Haemodialysis (HD) is often required for patients with end-stage renal disease. Arteriovenous fistulas (AVFs), a surgical procedure connecting an artery to a vein, are the preferred vascular access for HD due to their durability and lower complication rates. The aim of AVFs is to promote vein remodelling to accommodate increased blood flow needed for dialysis. However, many AVFs fail to mature properly, making them unsuitable for dialysis. Successful maturation requires remodelling, resulting in an increased luminal diameter and thickened walls to support the increased blood flow. After AVF creation, haemodynamic changes due to increased blood flow on the venous side of the AVF initiate a cascade of events that, when successful, lead to the proper maturation of the AVF, making it suitable for cannulation. In this process, endothelial cells play a crucial role since they are in direct contact with the frictional forces exerted by the blood, known as shear stress. Patients requiring HD often have other conditions that increase the burden of senescent cells, such as ageing, diabetes and hypertension. These senescent cells are characterized by irreversible growth arrest and the secretion of pro-inflammatory and pro-thrombotic factors, collectively known as the senescence-associated secretory phenotype (SASP). This accumulation can impair vascular function by promoting inflammation, reducing vasodilatation, and increasing thrombosis risk, thus hindering proper AVF maturation and function. This review explores the contribution of senescent endothelial cells to AVF maturation and explores potential therapeutic strategies to alleviate the effects of senescent cell accumulation, aiming to improve AVF maturation rates.

Non-resolving inflammation is the underpinning of several prevalent diseases including atherosclerosis. Understanding new mechanisms to promote the resolution of inflammation in atherosclerosis are of interest. Resolution is mediated by resolvins, including Resolvin D1 (RvD1). We recently showed that RvD1 prevents lesional necrosis in Ldlr -/- mice. However, the mechanisms underlying RvD1’s protective actions remain unknown. In this regard, the accumulation of senescent cells (SCs) was recently shown to promote necrotic core formation in Ldlr -/- mice. SCs are harmful because they possess a pro-inflammatory and proteolytic phenotype called the senescence associated secretory phenotype (SASP). Because RvD1 decreased lesional necrosis, we questioned whether RvD1’s actions were through limiting SCs in plaques. First, we found that human symptomatic plaques had significantly less RvD1 and significantly more SCs (quantified by positive p16 INK4A immunofluorescence staining) compared with asymptomatic plaques. To prove causation, we administered RvD1 to Ldlr -/- mice during advanced atherosclerosis and observed a significant decrease in lesional SCs and necrosis compared with vehicle controls. Mechanistically, we found that RvD1 significantly decreased the SASP as well as beta-galactosidase and p16 INK4A in WI-38 senescent cells. Importantly, a major component of the SASP is ADAM17, which is an enzyme that cleaves (and thus inactivates) a critical efferocytosis receptor on macrophages called MerTK. Because SCs are not cleared in plaques we questioned whether the SASP deranges efferocytosis mechanisms via their ability to cleave MerTK on macrophages. To investigate whether MerTK cleavage deranges SC clearance in plaques, we transplanted bone marrow from wild type (WT) or MerTK cleavage resistant (Mertk CR ) mice into Ldlr -/- mice, and quantified the levels of lesional p16 INK4A after 12 weeks of Western Diet feeding. Mertk CR mice had significantly less p16 INK4A levels and smaller necrotic cores compared with WT controls. Overall, we identified new functions for RvD1 and uncovered, for the first time, an endogenous mechanism for SC clearance. Our findings may provide new therapeutic strategies to treat atherosclerosis.

ABSTRACTCellular senescence is an aging‐related mechanism characterized by cell cycle arrest, macromolecular alterations, and a senescence‐associated secretory phenotype (SASP). Recent preclinical trials established that senolytic drugs, which target survival mechanisms of senescent cells, can effectively intervene in age‐related pathologies. In contrast, senomorphic agents inhibiting SASP expression while preserving the survival of senescent cells have received relatively less attention, with potential benefits hitherto underexplored. By revisiting a previously screened natural product library, which enabled the discovery of procyanidin C1 (PCC1), we noticed pyrroloquinoline quinone (PQQ), a redox cofactor that displayed remarkable potential in serving as a senomorphic agent. In vitro data suggested that PQQ downregulated the full spectrum expression of the SASP, a capacity observed in several stromal cell lines. Proteomics data supported that PQQ directly targets the intracellular protein HSPA8, interference with which disturbs downstream signaling and expression of the SASP. PQQ restrains cancer cell malignancy conferred by senescent stromal cells in culture while reducing drug resistance when combined with chemotherapy in anticancer regimens. In preclinical trials, PQQ alleviates pathological symptoms by preventing organ degeneration in naturally aged mice while reserving senescent cells in the tissue microenvironment. Together, our study supports the feasibility of exploiting a redox‐active quinone molecule with senomorphic capacity to achieve geroprotective effects by modulating the SASP, thus providing proof‐of‐concept evidence for future exploration of natural antioxidant agents to delay aging and ameliorate age‐related conditions. Prospective efforts are warranted to determine long‐term outcomes and the potential of PQQ for the intervention of geriatric syndromes in clinical settings.

Introduction: Senescent cells have emerged as bona fide drivers of ageing and age-related cardiovascular disease, with senescent cells accumulating in the aged heart and following damage/injury. We have shown that the removal of senescent cells using senolytics can rejuvenate the regenerative capacity of the aged heart. Aim: To investigate the effects of cell senescence and the action of the senolytics, Dasatinib (D) and Quercetin (Q) on human iPSC-derived cardiomyocyte survival and cell cycle, and endothelial cell survival, cell cycle, migration and tube formation in vitro. Methods and Results: We developed a transwell insert co-culture stress-induced premature senescence human cell model system to test the effects of senolytics D+Q in vitro. Co-culture of iPSC-derived cardiomyocytes (iPSC-CMs) with senescent cardiac stromal progenitor cells (senCPCs) led to decreased number and DNA-synthesising activity of iPSC-CMs. Treatment with senolytics D+Q led to the elimination of senCPCs in the co-culture and the rescue of iPSC-CM number and DNA synthesis. Treatment of HUVECs with senCPC conditioned media decreased HUVEC number, cell cycle activity, migration, and tube formation. Treatment of HUVECs with D+Q conditioned media rescued HUVEC number, migration and tube formation. Next, we investigated the effects of co-culture of senescent HUVECs (senHUVECs) with HUVECs and showed decreased HUVEC number and DNA synthesis. Treatment with senolytics D+Q led to the elimination of senHUVECs in the co-culture and ameliorated HUVEC number, but not DNA synthesis. Treatment of HUVECs with conditioned media from senHUVECs led to decreased HUVEC migration and tube formation. Treatment of HUVECs with D+Q conditioned media improved HUVEC tube formation but not migration. Luminex analysis of the conditioned media from iPSC-CM and HUVEC co-cultures revealed upregulation of senescence-associated secretory phenotype (SASP) factors, but the level of SASP factors was reduced with the application of D+Q. Conclusion: Senescent cell removal by senolytics D+Q shows therapeutic potential in rejuvenating the reparative activity of human cardiomyocytes and endothelial cells. These results open the path to further studies on using senolytic therapy in age-related cardiac deterioration and rejuvenation. Potential impact of the findings: Senescent cells and their SASP present a promising therapeutic target to rejuvenate the heart’s reparative potential. Clinical trials using senolytics D+Q are already underway and thus far have shown promising results. Further pre-clinical studies are warranted for evidence-based clinical trials using senolytics in age-related cardiovascular diseases.

Skin diseases affect nearly one quarter of the global population, with prevalence rising sharply among older adults. By 2050, the number of individuals over 60 years will double, making age-related dermatological conditions an increasing public health concern. A central process underlying aging is cellular senescence, a stable form of growth arrest induced by diverse stressors, including DNA damage, telomere attrition, and oncogenic signaling. Senescent keratinocytes, fibroblasts, melanocytes, and immune cells accumulate in the skin with age and secrete a pro-inflammatory senescence-associated secretory phenotype (SASP) that actively shapes the cutaneous immune landscape. Although senescence can promote tissue repair and tumour suppression, the persistence of senescent cells drives inflammation, impairs immunity function, and contributes to pathology, a concept now termed senopathy. In this review, we first examine the crosstalk between senescent stromal and immune cells in human skin. Then we discuss how SASP from senescent fibroblasts inhibits the function of resident T cell, while senescent T cells adopt a paradoxical state, hyperfunctional yet non-proliferative, that can accelerate tissue damage. We further highlight the immune evasion strategies which enable senescent cells to persist and drive inflammaging. Insights from patients with Familial Melanoma Syndrome (germline CDKN2A mutations) illustrate how defective senescence pathways across multiple cell types impair cutaneous immunosurveillance and increase melanoma risk. Finally, we explore evidence for stromal- and immune-mediated cutaneous senopathies, including psoriasis, lupus, vitiligo, and leishmaniasis, where senescent cells actively drive the diseases’ progression. Based on the analysis, we propose that the skin represents a powerful and accessible model for investigating the interplay between senescent immune and non-immune cells across the lifespan, with therapeutic implications for aging and age-related pathologies.

Cellular senescence is the process of the permanent proliferative arrest of cells in response to various inducers. It is accompanied by typical morphological changes, in addition to the secretion of bioactive molecules, including proinflammatory cytokines and chemokines [known as the senescence-associated secretory phenotype (SASP)]. Thus, senescent cells may affect their local environment and induce a so-called ‘bystander’ senescence through the state of SASP. The phenotypes of senescent cells are determined by the type of agent inducing cellular stress and the cell lineages. To characterise the phenotypes of senescent cancer cells, two murine cell lines were employed in the present study: TC-1 and B16F10 (B16) cells. Two distinct senescence inductors were used: Chemotherapeutic agent docetaxel (DTX) and a combination of immunomodulatory cytokines, including interferon γ (IFNγ) and tumour necrosis factor α (TNFα). It was demonstrated that DTX induced senescence in TC-1 and B16 tumour cell lines, which was demonstrated by growth arrest, positive β-galactosidase staining, increased p21Waf1 (p21) expression and the typical SASP capable of inducing a ‘bystander’ senescence. By contrast, treatment with a combination of T helper cell 1 cytokines, IFNγ and TNFα, induced proliferation arrest only in B16 cells. Despite the presence of certain characteristic features resembling senescent cells (proliferation arrest, morphological changes and increased p21 expression), these cells were able to form tumours in vivo and started to proliferate upon cytokine withdrawal. In addition, B16 cells were not able to induce a ‘bystander’ senescence. In summary, the present study described cell line- and treatment- associated differences in the phenotypes of senescent cells that may be relevant in optimization of cancer chemo- and immunotherapy.

Getting Ready for Wound Certification: Assessing and Managing Lower Extremity Venous Disease.

We tested the hypothesis that 1,25‐dihydroxyvitamin D3[1α,25(OH)2D3] has antiaging effects via upregulating nuclear factor (erythroid‐derived 2)‐like 2 (Nrf2), reducing reactive oxygen species (ROS), decreasing DNA damage, reducing p16/Rb and p53/p21 signaling, increasing cell proliferation, and reducing cellular senescence and the senescence‐associated secretory phenotype (SASP). We demonstrated that 1,25(OH)2D3‐deficient [1α(OH)ase−/−] mice survived on average for only 3 months. Increased tissue oxidative stress and DNA damage, downregulated Bmi1 and upregulated p16, p53 and p21 expression levels, reduced cell proliferation, and induced cell senescence and the senescence‐associated secretory phenotype (SASP) were observed. Supplementation of 1α(OH)ase−/− mice with dietary calcium and phosphate, which normalized serum calcium and phosphorus, prolonged their average lifespan to more than 8 months with reduced oxidative stress and cellular senescence and SASP. However, supplementation with exogenous 1,25(OH)2D3 or with combined calcium/phosphate and the antioxidant N‐acetyl‐l‐cysteine prolonged their average lifespan to more than 16 months and nearly 14 months, respectively, largely rescuing the aging phenotypes. We demonstrated that 1,25(OH)2D3exerted an antioxidant role by transcriptional regulation of Nrf2 via the vitamin D receptor (VDR). Homozygous ablation of p16 or heterozygous ablation of p53 prolonged the average lifespan of 1α(OH)ase−/− mice on the normal diet from 3 to 6 months by enhancing cell proliferative ability and reducing cell senescence or apoptosis. This study suggests that 1,25(OH)2D3 plays a role in delaying aging by upregulating Nrf2, inhibiting oxidative stress and DNA damage，inactivating p53‐p21 and p16‐Rb signaling pathways, and inhibiting cell senescence and SASP.

Background: Invasive lobular carcinoma (ILC) is the second most common histological breast cancer subtype; however, little is known about its tumor microenvironment (TME). Here, we aimed to identify and to characterise ILC subgroups based on TME heterogeneity by combining spatial transcriptomics (ST) and single cell RNA-sequencing (scRNA-seq). Methods: We performed ST (Visium 10x Genomics) on frozen tumor samples from 41 primary hormone receptor positive (HR+), HER2-negative (HER2-) ILCs. Information coming from the morphological annotation of ST slides and the gene expression-based clustering of ST spots was integrated and used as input for a patient level classification (using intNMF algorithm). Subgroups of patients were annotated using morphology, gene set enrichment analysis and ST deconvolution based on scRNA-seq data (CARD software). SCAN-B bulk RNA-seq dataset was used as validation set (HR+, HER2- ILC samples, n = 853). Results: Four subgroups of patients were identified: proliferative (P, n = 12, enriched in tumor cells and proliferation-related pathways), normal-stroma enriched (NSE, n = 10, enriched in fibroblasts, stroma-related pathways and showing an higher level of colocalization between invasive and in situ carcinoma), metabolic (M, n = 9, enriched in metabolic related-pathways) and metabolic-immune enriched (MIE, n = 10, enriched in adipocytes, metabolic and immune-related pathways). Deconvolution of ST spots with single cell data revealed matching results with morphology (in terms of cell types). We observed an enrichment of myofibroblasts in NSE, an enrichment of cancer epithelial in P and, interestingly, an enrichment of endothelial cells in M subgroup (fdr < 0.25). MIE was characterized by perivascular like-endothelial cells and myeloid cells (monocytes and macrophages, fdr < 0.25). Of note, at the level of the ST slide, myeloid cells in MIE subgroup were more present in the adipose tissue compartment (fdr < 0.25) compared to the rest of the slide. NSE subgroup showed a trend for association with better prognosis. Gene signature-based assignment identified the same four subgroups in SCAN-B: GSEA analysis and comparisons among the four subgroups showed matching results between our dataset and SCAN-B. Of note, in SCAN-B, we observed differences in prognosis among the subgroups, with NSE showing better, M and MIE intermediate and P worse prognosis for overall survival and relapse free interval (p < 0.01). Conclusions: Four subgroups of ILC based on TME heterogeneity and showing differences in prognosis were identified and validated in external cohort. Of note, two of these groups were related to metabolism, highlighting the value of such process in ILC biology. Importantly, myeloid cells were the predominant immune cell types in ILC, and they showed a specific compartmentalization inside the TME. Further validation is needed. Citation Format: Matteo Serra, Andreas Papagiannis, Mattia Rediti, Frederic Lifrange, Nicola Occelli, Laetitia Collet, Delphine Vincent, Ghizlane Rouas, Danai Fimereli, Ligia Craciun, Denis Larsimont, David Venet, Miikka Vikkula, Francois P. Duhoux, Françoise Rothé, Christos Sotiriou. Integrating spatial and single cell transcriptomics to identify and characterise biologically driven subgroups in invasive lobular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5066.