Stromal Compartment Research Articles

Abstract C109: Nanostring-based subtyping of pancreatic ductal adenocarcinoma is strongly influenced by the stromal compartment

Abstract The prognostic significance of basal-like and classical subtypes of pancreatic ductal adenocarcinoma (PDAC) is well-established, and ongoing clinical trials are poised to validate the predictive utility of such subtypes in guiding first-line treatment decision-making. While the Purity-independent (PurIST)-based subtyping strategy provides a method for PDAC subtype determination based on the Nanostring gene expression quantification platform, the utility of this platform has not been tested in a real-world patient setting. Here, we leverage a retrospective dataset of patients with resectable PDAC (n=299) that received Nanostring sequencing in combination with comprehensive clinical metadata curation and histopathological analysis. All sequencing analyses were performed using formalin-fixed paraffin-embedded (FFPE) tumor whole sections. The proportion of basal-like cases (15%) was comparable with other resectable PDAC cohorts, and patients with basal-like tumors showed reduced overall survival (p=3.9e-4). Histopathological quantification of epithelial, stromal and normal components in each sample revealed significantly (p=0.007) reduced classical gene expression in stroma-high samples. Out of 29 patients with repeat sampling from different FFPE blocks derived from the same tumor and surgery, 31% of tumors received discordant subtypes calls and increases in the amount of stroma sequenced between two samples from the same tumor was negatively correlated with expression of classical subtype genes (p=0.02). Taken together, these data demonstrate the presence of intra-tumoral subtype heterogeneity, highlight the quantity of stroma sequenced as a major contributing factor in PurIST-based subtype determination and generate insight into the reproducibility of this subtyping strategy in a real-world clinical setting. Citation Format: James T. Topham, Steve E. Kalloger, Joanna M. Karasinska, Jenny E. Chu, Hassan Ali, Dongxia Gao, Christine Chow, Andrew Metcalfe, Jonathan M. Loree, David F. Schaeffer, Daniel J. Renouf. Nanostring-based subtyping of pancreatic ductal adenocarcinoma is strongly influenced by the stromal compartment [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C109.

Abstract C030: Serum levels and tumor tissue organization of decorin in pancreatic cancer: Links to chemoresistance and disease aggressiveness

Abstract Pancreatic cancer has nearly paralleling incidence and mortality rates. Only 15-20% of the tumors are resectable at presentation, and for the majority of patients the situation is palliative. Treatment options are limited to standard chemotherapy, and tumor response is often transient. The longitudinal observational clinical study Chemotherapy, Host response And Molecular dynamics in Periampullary cancer (CHAMP) aims to contribute with new insights on this devastating disease through longitudinal blood sampling during adjuvant and palliative chemotherapy and comprehensive analysis of tissue from resected as well as postmortem sampled tumor tissue. In the present study, encompassing the first 100 patients enrolled in the CHAMP study, 92 serum proteins related to inflammation and cancer were analyzed by proximity extension assay in on-treatment blood samples from baseline to six months. The results revealed that the small leucin-rich proteoglycan decorin was among the top up-regulated proteins during chemotherapy. After three months of treatment, high levels of serum decorin were significantly associated with a shorter overall survival (log rank p = 0.005). Decorin expression was also assessed by immunohistochemistry in resected tumors from 25 patients and in postmortem sampled primary tumors and metastases from 9 patients. In the latter, particularly strong expression of decorin, both in the tumor cells and in the stroma, was denoted in cases with a short overall survival. While there was no significant correlation between decorin in the tumors and in serum at baseline, strong correlations were seen at one and three months (Spearman’s rho = 0.70 and 0.85, respectively). To further validate the potential prognostic value of decorin, immunohistochemical analysis was performed on tumors from a retrospective cohort of 104 resected tumors with long-term follow-up. Survival analysis showed that high decorin expression in both the stromal compartment and in tumor cells was associated with shorter overall survival (log rank p = 0.036 and 0.001, respectively). Multivariable cox regression analysis, adjusted for conventional prognostic factors, further confirmed that decorin was an independent factor of shorter overall survival (hazard ratio: 1.74, 95% confidence interval 1.10-2.75 for stromal decorin, and hazard ratio: 1.83, 95% confidence interval 1.04-3.21 for tumor-cell specific decorin). In conclusion, these findings suggest that decorin may be associated with chemotherapy resistance in pancreatic cancer. Future studies are encouraged to further explore the mechanistic basis for these observations, and to validate the potential utility of serum-decorin as a biomarker to monitor treatment response. Citation Format: Maja Svensson, Johan Ohlsson, Sofie Olsson Hau, Alexandra Petersson, Karin Jirström. Serum levels and tumor tissue organization of decorin in pancreatic cancer: Links to chemoresistance and disease aggressiveness [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C030.

Abstract C028: DKK3 in pancreatic cancer – Elucidating the roles of a double-edged sword

Abstract Pancreatic ductal adenocarcinoma (PDAC) remains a highly challenging disease with a poor prognosis due to high tumor heterogeneity, lack of diagnostic biomarkers and limited treatment options. Therefore, it is crucial to meticulously examine the intricate molecular mechanisms involved in the interplay between different cellular compartments in PDAC. We previously identified DKK3 (Dickkopf-3) as a novel factor promoting organ regeneration during pancreatitis and established a previously unknown link between DKK3 and the Wnt and Hedgehog signaling. Given that pancreatitis is a significant risk factor for pancreatic cancer, we were motivated to investigate the potential role of DKK3 in PDAC development. We utilized PDAC mouse models driven by LSL-KrasG12D/+; Ptf1aCre/+ with or without homozygous/heterozygous DKK3 deletion in both epithelial and stromal compartment and employed various in vitro and ex vivo systems to examine the stage and compartment-specific roles of DKK3 during PDAC progression. Through a comprehensive and time-resolved analysis of tumor characteristics, we discovered that the loss of DKK3 led to rapid oncogenic transformation, marked by a significant increase in metaplasia and desmoplasia starting at an early stage (10 weeks). Ex vivo acinar-to-ductal metaplasia assay and rescue experiments with wildtype and DKK3-null acinar cells revealed that secreted DKK3 operates as a tumor suppressive roadblock in PDAC-initiating steps. This advanced preneoplastic stage translated into accelerated progression toward invasive tumors at 36 weeks, shortened overall survival, and a higher liver metastasis incidence in the DKK3-null animals at the endpoint. Further characterization of isolated tumor cells underpinned our observations, revealing prominent mesenchymal features and enhanced migratory capacities upon loss of DKK3. Interestingly, DKK3-null PDACs exhibited a pronounced stroma remodeling with a myofibroblastic cancer-associated fibroblast-enriched environment, as well as an immunosuppressive stroma with abundant regulatory T cells and cytotoxic T cell depletion, as substantiated by coculture experiments. Intriguingly, while displaying similar phenotypic features to DKK3-null tumors, Dkk3+/- heterozygotes exhibited the most aggressive outcome. To further dissect this finding, we conducted a set of in silico approaches and immunostainings revealing persistent stromal DKK3 expression in DKK3-proficient PDACs. This suggested a possible dual role of DKK3 that could operate either as a friend or a foe depending on its spatiotemporal expression in PDAC. In line, in vitro systems demonstrated a distinct oncogenic effect of DKK3 on endpoint tumor cells, particularly promoting malignant cell migration. In summary, we propose that epithelial DKK3 exerts cell-autonomous tumor suppressive functions during PDAC onset, while stromal DKK3 acts as an oncogenic cue driving cancer progression. These findings highlight DKK3 as a promising biomarker and a novel therapeutic target for addressing the oncogenic dialog within PDAC tumors. Citation Format: Dharini Srinivasan, Frank Arnold, Elodie Roger, Anna Härle, Michael Melzer, Patrick Hermann, Lukas Perkhofer, Johann Gout, Alexander Kleger. DKK3 in pancreatic cancer – Elucidating the roles of a double-edged sword [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C028.

Proteolytic remodeling of 3D bioprinted tumor microenvironments

In native tissue, remodeling of the pericellular space is essential for cellular activities and is mediated by tightly regulated proteases. Protease activity is dysregulated in many diseases, including many forms of cancer. Increased proteolytic activity is directly linked to tumor invasion into stroma, metastasis, and angiogenesis as well as all other hallmarks of cancer. Here we show a strategy for 3D bioprinting of breast cancer models using well-defined protease degradable hydrogels that can facilitate exploration of the multifaceted roles of proteolytic extracellular matrix remodeling in tumor progression. We designed a set of bicyclo[6.1.0]nonyne functionalized hyaluronan (HA)-based bioinks cross-linked by azide-modified poly(ethylene glycol) (PEG) or matrix metalloproteinase (MMP) degradable azide-functionalized peptides. Bioprinted structures combining PEG and peptide-based hydrogels were proteolytically degraded with spatial selectivity, leaving non-degradable features intact. Bioprinting of tumor-mimicking microenvironments using bioinks comprising human breast cancer cells (MCF-7) and fibroblast in hydrogels with different susceptibilities to proteolytic degradation shows that MCF-7 proliferation and spheroid size were significantly increased in protease degradable hydrogel compartments, but only in the presence of fibroblasts. In the absence of fibroblasts in the stromal compartment, cancer cell proliferation was reduced and did not differ between degradable and nondegradable hydrogels. The interactions between spatially separated fibroblasts and MCF-7 cells consequently resulted in protease-mediated remodeling of the bioprinted structures and a significant increase in cancer cell spheroid size, highlighting the close interplay between cancer cells and stromal cells in the tumor microenvironment and the influence of proteases in tumor progression.

Subclassification of B-acute lymphoblastic leukemia according to age, immunophenotype and microenvironment, predicts MRD risk in Mexican children from vulnerable regions.

The decisive key to disease-free survival in B-cell precursor acute lymphoblastic leukemia in children, is the combination of diagnostic timeliness and treatment efficacy, guided by accurate patient risk stratification. Implementation of standardized and high-precision diagnostic/prognostic systems is particularly important in the most marginalized geographic areas in Mexico, where high numbers of the pediatric population resides and the highest relapse and early death rates due to acute leukemias are recorded even in those cases diagnosed as standard risk. By using a multidimensional and integrated analysis of the immunophenotype of leukemic cells, the immunological context and the tumor microenvironment, this study aim to capture the snapshot of acute leukemia at disease debut of a cohort of Mexican children from vulnerable regions in Puebla, Oaxaca and Tlaxcala and its potential use in risk stratification. Our findings highlight the existence of a distinct profile of ProB-ALL in children older than 10 years, which is associated with a six-fold increase in the risk of developing measurable residual disease (MRD). Along with the absence of CD34+ seminal cells for normal hematopoiesis, this ProB-ALL subtype exhibited several characteristics related to poor prognosis, including the high expression level of myeloid lineage markers such as MPO and CD33, as well as upregulation of CD19, CD34, CD24, CD20 and nuTdT. In contrast, it showed a trend towards decreased expression of CD9, CD81, CD123, CD13, CD15 and CD21. Of note, the mesenchymal stromal cell compartment constituting their leukemic niche in the bone marrow, displayed characteristics of potential suppressive microenvironment, such as the expression of Gal9 and IDO1, and the absence of the chemokine CXCL11. Accordingly, adaptive immunity components were poorly represented. Taken together, our results suggest, for the first time, that a biologically distinct subtype of ProB-ALL emerges in vulnerable adolescents, with a high risk of developing MRD. Rigorous research on potential enhancing factors, environmental or lifestyle, is crucial for its detection and prevention. The use of the reported profile for early risk stratification is suggested.

Spatial proteomic profiling of tumor and stromal compartments in non-small-cell lung cancer identifies signatures associated with overall survival.

Non-small-cell lung carcinoma (NSCLC) is the most prevalent and lethal form of lung cancer. The need for biomarker-informed stratification of targeted therapies has underpinned the need to uncover the underlying properties of the tumor microenvironment (TME) through high-plex quantitative assays. In this study, we profiled resected NSCLC tissues from 102 patients by targeted spatial proteomics of 78 proteins across tumor, immune activation, immune cell typing, immune-oncology, drug targets, cell death and PI3K/AKT modules to identify the tumor and stromal signatures associated with overall survival (OS). Survival analysis revealed that stromal CD56 (HR = 0.384, P = 0.06) and tumoral TIM3 (HR = 0.703, P = 0.05) were associated with better survival in univariate Cox models. In contrast, after adjusting for stage, BCLXL (HR = 2.093, P = 0.02) and cleaved caspase 9 (HR = 1.575, P = 0.1) negatively influenced survival. Delta testing indicated the protective effect of TIM-3 (HR = 0.614, P = 0.04) on OS. In multivariate analysis, CD56 (HR = 0.172, P = 0.001) was associated with better survival in the stroma, while B7.H3 (HR = 1.72, P = 0.008) was linked to poorer survival in the tumor. Deciphering the TME using high-plex spatially resolved methods is giving us new insights into compartmentalised tumor and stromal protein signatures associated with clinical endpoints in NSCLC.

Avaliação da remodelação tecidual prostática em ratos expostos ao bisfenol A e aos fitoestrógenos genisteína e indol-3-carbinol durante o período perinatal

ABSTRACT: Bisphenol A (BPA) is a compound known for its direct action on the prostate. Prostatic morphogenesis is a critical period when interference by any compound could permanently damage the organ. As such, the present study evaluated the morphological aspects resulting from gestational and lactational administration of BPA, indole-3-carbinol (I3C) and genistein (GEN) in prepubescent male rats. Pregnant Sprague Dawley females were allocated into 4 experimental groups and received the following: C: Control (no treatment); B: BPA (10 µg/Kg); BG: BPA+GEN (5 mg/Kg); BI: BPA+I3C (20 mg/Kg) from gestation day (GD) 17 to postnatal day (PND) 21. After euthanasia on PND22, the prostate was collected and processed. When administered alone, BPA reduced the stromal compartment when compared to group C (P = 0.039). This decline was reversed in the groups submitted to GEN (P = 0.019) or I3C (P = 0.017). The groups treated with BPA (P < 0.0001) and the phytoestrogens (P < 0.0001) exhibited decreased epithelial height in relation to the control group. These changes were observed in stereological and morphometric analyses, but not in fractal analysis (P = 0.569). The area occupied by collagen increased in groups treated with BPA (P < 0.0001) and phytoestrogens (P < 0.0001) in relation to controls, while collagen distribution values were higher in all the treated groups (P < 0.0001), according to fractal analysis. Thus, BPA induced prostrate stroma remodeling with no influence from the phytoestrogens, which may affect glandular development and cause histopathological changes in adulthood.

B-cell infiltration is associated with survival outcomes following programmed cell death protein 1 inhibition in head and neck squamous cell carcinoma

Programmed cell death protein 1 (PD-1) axis blockade has become the mainstay in the treatment of recurrent and/or metastatic (R/M) head and neck squamous cell cancer (HNSCC). Programmed death-ligand 1 (PD-L1) is the only approved biomarker for patient selection; however, response rate is limited even among high expressors. Our primary objective was to investigate the association of immune cell-related biomarkers in the tumor and tumor microenvironment with PD-1 checkpoint inhibitors' outcomes in patients with R/M HNSCC. NCT03652142 was a prospective study in nivolumab-treated platinum-refractory R/M HNSCC, aiming to evaluate biomarkers of response to treatment. Tumor biopsies and blood samples were collected from 60 patients at baseline, post-treatment, and at progression. Immune cells in the tumor and stromal compartments were quantified by immunofluorescence using a five-protein panel (CD3, CD8, CD20, FoxP3, cytokeratin). Tertiary lymphoid structures (TLSs), PD-L1 expression, and peripheral blood immune cell composition were also evaluated for associations with outcome. Our findings were validated by gene set enrichment analysis (GSEA) messenger RNA in situ expression data from the same patients, for B-cell- and TLS-associated genes. High pre-treatment density of stromal B cells was associated with prolonged progression-free survival (PFS) (P= 0.011). This result was validated by GSEA, as stromal enrichment with B-cell-associated genes showed association with response to nivolumab. PD-L1 positivity combined with high B-cell counts in stroma defined a subgroup with significantly longer PFS and overall survival (P= 0.013 and P= 0.0028, respectively). Increased B cells in pre-treatment HNSCC biopsy samples correlate with prolonged benefit from PD-1-based immunotherapy and could further enhance the predictive value of PD-L1 expression.

Electroanalytical Immunotool to Determine Matricellular Protein Periostin, a Stromal Biomarker of Prognosis in Colorectal Cancer

AbstractTumor‐associated stroma biomarkers are emerging as key signaling molecules of metastasis in colorectal cancer (CRC). In this sense, periostin (POSTN), a protein of the extracellular matrix from the stromal compartment, is envisioned as a potential stromal prognostic biomarker, which facilitates the application of pertinent treatments. In this work, we report an easy‐to‐handle amperometric sandwich‐based immunosensing strategy for the determination of POSTN involving commercial magnetic microparticles (MBs), disposable carbon electrodes, and the horseradish peroxidase (HRP)/H2O2/hydroquinone (HQ) electrochemical system. The method allowed a dynamic linear range between 0.47 and 25 ng mL−1 and a limit of detection, LOD, of 0.14 ng mL−1 compatible with clinical demands. The method was applied to the analysis of POSTN in a variety of cancer‐related real bio‐scenarios including cell extracts and secretomes from CRC cells, and plasma from CRC patients at different stages. The potentials of this firstly developed MBs‐assisted immunoplatform are justified by distinguishing between metastatic abilities of cultured CRC cells through the analysis of their extracts and secretomes, and by differentiating between healthy controls and CRC patients in just 60 min. Therefore, the developed immunoplatform can be envisaged as a novel and profitable tool for exploring the most uncharacterized but prospective tumor areas.

Spatial transcriptomic interrogation of the tumour-stroma boundary in a 3D engineered model of ameloblastoma

Stromal cells are key components of the tumour microenvironment (TME) and their incorporation into 3D engineered tumour-stroma models is essential for tumour mimicry. By engineering tumouroids with distinct tumour and stromal compartments, it has been possible to identify how gene expression of tumour cells is altered and influenced by the presence of different stromal cells. Ameloblastoma is a benign epithelial tumour of the jawbone. In engineered, multi-compartment tumouroids spatial transcriptomics revealed an upregulation of oncogenes in the ameloblastoma transcriptome where osteoblasts were present in the stromal compartment (bone stroma). Where a gingival fibroblast stroma was engineered, the ameloblastoma tumour transcriptome revealed increased matrix remodelling genes. This study provides evidence to show the stromal-specific effect on tumour behaviour and illustrates the importance of engineering biologically relevant stroma for engineered tumour models. Our novel results show that an engineered fibroblast stroma causes the upregulation of matrix remodelling genes in ameloblastoma which directly correlates to measured invasion in the model. In contrast the presence of a bone stroma increases the expression of oncogenes by ameloblastoma cells.

Exploration of cancer associated fibroblasts phenotypes in the tumor microenvironment of classical and pleomorphic Invasive Lobular Carcinoma.

Multiplex immunofluorescence were performed on formalin fixed paraffin embedded tissues using Opal-7 color kit. The antibodies used for phenotyping CAFs were Pan CK (AE1/AE3), CD45, A-SMA, FAP, S100, Thy-1 with optimized dilutions. The images were acquired and analyzed using Vectra 3.0 imaging system and InForm software respectively. We studied 19 different CAFs colocalized phenotypes in the tumor, stroma and overall tissue compartments between classic and pleomorphic ILC. Total A-SMA+, A-SMA+FAP+S100+ and A-SMA+S100+ CAFs demonstrated higher densities in classic ILC cases while FAP+S100+ and S-100+ CAFs were increased in the pleomorphic subtype samples. Our study explores multiple CAFs phenotypes between classical and pleomorphic ILC. We showed that CAFs subset differ between Classic ILC and Pleomorphic ILC. A-SMA CAFs are more prevalent in the TME of classic ILCs whereas Pleomorphic ILCs are dominated by CAFs without A-SMA expression. This also iterates the importance of exploring this particular type of breast carcinoma in more detail, paving the way for meaningful translational research.

Redox regulation in chloroplast thylakoid lumen: The pmf changes everything, again.

Photosynthesis is the foundation of life on Earth. However, if not well regulated, it can also generate excessive reactive oxygen species (ROS), which can cause photodamage. Regulation of photosynthesis is highly dynamic, responding to both environmental and metabolic cues, and occurs at many levels, from light capture to energy storage and metabolic processes. One general mechanism of regulation involves the reversible oxidation and reduction of protein thiol groups, which can affect the activity of enzymes and the stability of proteins. Such redox regulation has been well studied in stromal enzymes, but more recently, evidence has emerged of redox control of thylakoid lumenal enzymes. This review/hypothesis paper summarizes the latest research and discusses several open questions and challenges to achieving effective redox control in the lumen, focusing on the distinct environments and regulatory components of the thylakoid lumen, including the need to transport electrons across the thylakoid membrane, the effects of pH changes by the proton motive force (pmf) in the stromal and lumenal compartments, and the observed differences in redox states. These constraints suggest that activated oxygen species are likely to be major regulatory contributors to lumenal thiol redox regulation, with key components and processes yet to be discovered.

Histological investigation in aging male and female gerbil prostates after prenatal exposure to pequi (Caryocar brasiliense Cambess) oil and 17α-ethinylestradiol

ABSTRACT The female prostate, also known as Skene’s gland, is present in both humans and rodents. Prenatal exposure to ethinylestradiol (EE2), a synthetic estrogen found in oral contraceptives, induces pormotes neoplasic prostate lesions in gerbils (Meriones unguiculatus). Conversely, pequi oil (Pe), extracted from the Brazilian Cerrado fruit, has antioxidant, anti-inflammatory, and anticancer properties, mitigates risks associated with chronic diseases related to lifestyle and aging. This study evaluates the impact of prenatal exposure to Pe (300 mg/kg) on senile gerbil offspring’s male and female prostates under normal conditions and EE2 exposure (15 μg/kg/day). Histological and morphometric analyses revealed that Pe reduced male body weight and prostate epithelial height, along with a thinner muscle layer. In females, EE2 exposure reduced prostatic weight, while Pe exposure lowered epithelial height and the relative stromal compartment volume, increasing the muscle layer. Pequi oil holds potential in mitigating alterations induced by exposure to the endocrine disruptor EE2.

Lymph node stromal cell responses to perinatal T cell waves, a temporal atlas.

The perinatal period is a critical time window in establishing T cell tolerance. Regulatory T cells (Tregs) made during the first 2 wk of life are key drivers of perinatal tolerance induction, but how these cells are generated and operate has not been established. To elucidate the unique environment murine perinatal Tregs encounter within the lymph nodes (LNs) as they first emerge from the thymus, and how it evolves over the succeeding days, we employed single-cell RNA sequencing to generate an atlas of the early LN niche. A highly dynamic picture emerged, the stromal cell compartment showing the most striking changes and putative interactions with other LN cell compartments. In particular, LN stromal cells showed increasing potential for lymphocyte interactions with age. Analogous studies on mice lacking α:β T cells or enriched for autoreactive α:β T cells revealed an acute stromal cell response to α:β T cell dysfunction, largely reflecting dysregulation of Tregs. Punctual ablation of perinatal Tregs induced stromal cell activation that was dependent on both interferon-gamma signaling and activation of conventional CD4+ T cells. These findings elucidate some of the earliest cellular and molecular events in perinatal induction of T cell tolerance, providing a framework for future explorations.

Abstract C168: Prognostic value of tumor microenvironment (TME) features in advanced, EGFR-mutant non-small cell lung cancer (NSCLC)

Abstract Background: Prognostic role of tumor microenvironment (TME) features, although well-studied in the context of immunotherapy responses, remains unclear for oncogene-driven non-small cell lung cancer (NSCLC). Here, we interrogate the impact of TME features on survival outcomes of patients with advanced EGFR-mutant NSCLC. Methods: A single-center, retrospective study was carried out on patients with advanced EGFR-mutant (exon 19 deletion/L858R) NSCLC who underwent first-line targeted therapy or chemotherapy. Clinical, radiological, molecular, pathological data were collected. Hematoxylin-eosin diagnostic tissue slides, digitalized at 20x magnification, were analyzed by an expert pathologist and a medical oncologist, blinded to clinical data, to describe TME. Tumor-infiltrating lymphocytes (TILs), excluding polymorphonucleates, were assessed in the stromal compartment as positive (+) or negative (-), with positivity threshold of 10% of stromal tissue area. Fibrosis, necrosis, tertiary lymphoid structures (TLSs) and lymphangitis were assessed with a positivity threshold of 1%. Progression free survival (PFS) and overall survival (OS) were calculated from the start of first-line therapy and compared by log-rank test. Multivariable analysis by Cox regression model for OS included age, gender, PS-ECOG, number (n) of metastatic sites, presence of brain metastases and TME features. Results: A total of 118 patients were included: 52 treated with first-line osimertinib, 47 with gefitinib/erlotinib, 19 with platinum-based chemotherapy. The patient population included 84 (71%) females, 77 (65%) never smokers. Median age was 64 years (range 34-91), median n of metastatic sites was 2, PS ECOG was <2 in 91 (77%) and brain metastases were present in 48 (41%) cases. Most common biopsy site was lung (70%). Fibrosis, TILs, lymphangitis, necrosis, TLSs were found in 76/95 (80%), 34/93 (37%), 35/95 (37%), 28/94 (30%), and 12/92 (13%) of evaluable samples, respectively. After a median follow-up of 50.3 months (m), median OS was 35.2 (95%CI 27.8-41.6) m in the overall population; 23.1 (95%CI 18.2-39.3) vs 36.3 (95%CI 32.1-51.3) m (p=0.007) in necrosis+ vs -; 27.7 (95%CI 26.6- not reached, NR) vs 35.5 (95% CI 27.8-48.5) m (p=0.85) in TILs+ vs TILs-; 38.1 (95%CI 36.6-NR) vs 32.1 (95%CI 27-44.7) m (p=0.94) in TLS+ vs TLS-. Nine (26%) TILs+ tumors were also necrosis+. Necrosis (HR 2.05, p=0.026) and PS>1 (HR 2.52, p<0.01) were independently associated with worse OS in the multivariable analysis. In necrosis+ vs -, median PFS to first-line was 11.4 (95%CI 3,6-NR) vs 11.2 (95%CI 9.3-NR) m in osimertinib (p=0.67) and 7.8 (95%CI 5.4-NR) vs 13 (95%CI 8.7-20.1) in erlotinib/gefitinib group (p=0.84). Conclusions: Necrosis emerges as a negative prognostic factor for EGFR-mutant NSCLC. The role of osimertinib in counteracting the impact of necrosis should be elucidated on a larger scale to yield more substantial data. Further analyses will be performed on patients treated with anti-angiogenic agents. Citation Format: Lodovica Zullo, Maria Rosa Ghigna, Wael Salem Zrafi, Arianna Marinello, Damien Vasseur, Maxime Frelaut, Pamela Abdayem, Marco Tagliamento, Pernelle Lavaud, Anas Gazzah, Jean-Yves Scoazec, Jordi Remon, Fabrice Barlesi, Benjamin Besse, David Planchard, Mihaela Aldea. Prognostic value of tumor microenvironment (TME) features in advanced, EGFR-mutant non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C168.

Testing the Limits of Endogenous Thymic Regeneration after HCT Conditioning

T cell reconstitution following allogeneic hematopoietic cell transplantation (allo-HCT), recently emerging as a major predictor of clinical outcomes, is critically dependent upon thymic function. Previous clinical studies have implicated a link between thymic function pre-transplant to patient outcomes post-bone marrow transplantation. Thymic function is primarily driven by two functionally distinct groups of thymic epithelial cells (TECs) located in the cortex (cTECs) and the medulla (mTECs). These two stromal compartments contribute to the various stages of T cell development, culminating in the generation of a broad repertoire of self-tolerant T cells leaving the thymus and emigrating into the periphery. However, despite the thymus' crucial role in generating a broad array of T cells, the organ is highly sensitive to damage. Condition regiments required for successful HCT, such as ionizing radiation, have a profound damaging effect on the thymus. Although the thymus is sensitive to damaging agents, it also has a remarkable self-repair capacity through mechanisms that are still incompletely understood; and the organ's regenerative capacity also declines with age. Moreover, the specific thymic epithelial subpopulation that contributes to thymus regeneration remains undefined. Therefore, understanding endogenous cellular mechanisms of thymus regeneration, and limitations to its repair, will be crucial for creating therapies to boost thymic function. Although thymic function was restored to pre-conditioning levels after one round of total body irradiation (TBI), and multiple rounds of corticosteroid (Dexamethasone) damage saw no decline in its restorative ability, thymic regeneration was severely reduced upon subsequent rounds of ionizing radiation damage (Fig. 1A). A phenomenon that one may expect to see in the small subset of patients that need to undergo a second HCT. Using single cell RNA sequencing of highly purified TECs at multiple consecutive days after TBI, we identified that shortly after damage the TEC compartment contracts into distinct TEC subpopulations, including a previously reported CD104 expressing mTEC1 subpopulation implicated to provide pre-cursor support to the thymic medulla (Borstein 2018 Nature). To further understand the fundamental differences in regeneration from these different damage modalities, we compared the regeneration of thymic epithelial cells after either dexamethasone (Dex) or TBI damage. Consistent with its progenitor role, we found a significant decrease in the mTEC1 subpopulation in mice damaged with TBI compared to those damaged with Dex. The striking distinction in the mTEC1 compartments between the damage models can potentially explain a key functional difference in how these two damage modalities differ in their regenerative kinetics (Fig 1B). Overall, the results of these studies implicate a potential contribution of a specific mTEC subset (mTEC1s) to modulating thymic regeneration kinetics, and that the thymus cannot withstand multiple rounds of irradiation. The identification of mTEC1s as crucial mediators of thymic regeneration can inform future work in designing thymic boosting therapies that induce this stromal population to accelerate thymus regeneration after HCT.

Mathematical modelling and deep learning algorithms to automate assessment of single and digitally multiplexed immunohistochemical stains in tumoural stroma

Whilst automated analysis of immunostains in pathology research has focused predominantly on the epithelial compartment, automated analysis of stains in the stromal compartment is challenging and therefore requires time-consuming pathological input and guidance to adjust to tissue morphometry as perceived by pathologists. This study aimed to develop a robust method to automate stromal stain analyses using 2 of the commonest stromal stains (SMA and desmin) employed in clinical pathology practice as examples. An effective computational method capable of automatically assessing and quantifying tumour-associated stromal stains was developed and applied on cores of colorectal cancer tissue microarrays. The methodology combines both mathematical models and deep learning techniques with the former requiring no training data and the latter as many inputs as possible. The novel mathematical model was used to produce a digital double marker overlay allowing for fast automated digital multiplex analysis of stromal stains. The results show that deep learning methodologies in combination with mathematical modelling allow for an accurate means of quantifying stromal stains whilst also opening up new possibilities of digital multiplex analyses.

Engineering a metastatic stroma directs the osteosarcoma tumour transcriptome in a spatially specific manner

The biological behaviour of the cancer cell is in part controlled by its microenvironment and in part by underlying genetics. The interaction of cancer cells with the surrounding stroma directs cancer progression. The most common primary malignant tumour of the bone, osteosarcoma, is a highly complex disease in terms of its progression, invasion, and metastasis. To better understand disease characteristics and develop accurate therapies for osteosarcoma, novel tissue models have been developed to systematically understand how facets of the tumour microenvironment (TME) can influence cancer cell behaviour. This study aims to understand how osteosarcoma invasion and metastasis is influenced by different types of stromal cells in the TME. The engineering of complex tumouroids, which segregate the tumour and stromal cells in specific 3D compartments, allows for the study of tumour-stroma interaction and invasion of cells between different compartments. Complex tumouroids comprised a central osteosarcoma tumour mass with different engineered stromal compartments, representative of the primary bone or metastatic lung. Spatial Transcriptomics (ST) unveiled that osteosarcoma cells upregulated metastatic genes such as IFI27 in the presence of a lung stromal compartment, whereas a bone stromal compartment resulted in the upregulated of oncogenes and drug resistance genes such as DUSP1 and HIF1A within the osteosarcoma compartment. Engineering a primary stromal cell compartment compared to a metastatic stromal cell compartment unveiled how the regulation of cell proliferation, invasion, extracellular matrix (ECM) remodelling, metastasis markers and tumour suppressor markers was altered in the osteosarcoma transcriptome.

Formation of an invasion-permissive matrix requires TGFβ/SNAIL1-regulated alternative splicing of fibronectin

BackgroundAs in most solid cancers, the emergence of cells with oncogenic mutations in the mammary epithelium alters the tissue homeostasis. Some soluble factors, such as TGFβ, potently modify the behavior of healthy stromal cells. A subpopulation of cancer-associated fibroblasts expressing a TGFβ target, the SNAIL1 transcription factor, display myofibroblastic abilities that rearrange the stromal architecture. Breast tumors with the presence of SNAIL1 in the stromal compartment, and with aligned extracellular fiber, are associated with poor survival prognoses.MethodsWe used deep RNA sequencing and biochemical techniques to study alternative splicing and human tumor databases to test for associations (correlation t-test) between SNAIL1 and fibronectin isoforms. Three-dimensional extracellular matrices generated from fibroblasts were used to study the mechanical properties and actions of the extracellular matrices on tumor cell and fibroblast behaviors. A metastatic mouse model of breast cancer was used to test the action of fibronectin isoforms on lung metastasis.ResultsIn silico studies showed that SNAIL1 correlates with the expression of the extra domain A (EDA)-containing (EDA+) fibronectin in advanced human breast cancer and other types of epithelial cancers. In TGFβ-activated fibroblasts, alternative splicing of fibronectin as well as of 500 other genes was modified by eliminating SNAIL1. Biochemical analyses demonstrated that SNAIL1 favors the inclusion of the EDA exon by modulating the activity of the SRSF1 splicing factor. Similar to Snai1 knockout fibroblasts, EDA- fibronectin fibroblasts produce an extracellular matrix that does not sustain TGFβ-induced fiber organization, rigidity, fibroblast activation, or tumor cell invasion. The presence of EDA+ fibronectin changes the action of metalloproteinases on fibronectin fibers. Critically, in an mouse orthotopic breast cancer model, the absence of the fibronectin EDA domain completely prevents lung metastasis.ConclusionsOur results support the requirement of EDA+ fibronectin in the generation of a metastasis permissive stromal architecture in breast cancers and its molecular control by SNAIL1. From a pharmacological point of view, specifically blocking EDA+ fibronectin deposition could be included in studies to reduce the formation of a pro-metastatic environment.

Single Cell Analysis of Bone Marrow Stromal Cells Shows That a Pro-Inflammatory and Stress Response Together with Polarized Differentiation of Stromal Elements Characterize the Bone Marrow Microenvironment during Multiple Myeloma Development

Current knowledge of the biological role of the non-hemopoietic components of the bone marrow (BM) niche to the pathogenesis of multiple myeloma (MM) is poorly understood. We utilized a murine model to interrogate niche forming cells early in the natural history of MM, at a precursor stage (MGUS/smoldering-like) in more detail. Studying interactions between the MM clone and the stromal components of the MM niche are challenging as they represent only approximately 0.2% of the total marrow content and are difficult to isolate. However, by isolating the stromal compartment of the BM by depleting hemopoietic cells via flow sorting combined with the use of single cell RNA sequencing (scRNA-seq) it is possible to perform an in-vivo analysis of the individual cellular components of the stromal microenvironment and to characterize their relative abundance, cellular differentiation state, and transcriptomic profile. To comprehensively assess changes in the stromal cells during the early stage of MM, 5TGM1 cells were intravenously injected into aged (6 month old) KaLwRij mice. KaLwRij mice without MM were used as controls. Stromal cells were isolated following tumor engraftment in the marrow at a precursor stage and enriched as previously described (Baryawno et al, Cell 2019). scRNA-seq was performed on 5 libraries of cells using the 10X Genomics Chromium platform. All data analysis was performed in R. After filtering out low quality cells, 45,030 cells remained for analysis. The 5 libraries were integrated with Seurat and run through a standard clustering workflow involving log-normalization, finding variable features, dimensional reduction, and clustering. Cell type annotation for each cluster was performed manually using established markers for both the stromal and immune compartments. After excluding immune cell populations, 14,219 cells remained. Select subpopulations were re-clustered by isolating the cell types of interest and running them through separate standard clustering workflows. For each population of interest, differentially expressed genes were determined using the Wilcox rank sum test in Seurat, trajectory analysis and pseudotime were computed using Monocle3, and enriched gene sets were computed using GSEAPreranked. We identified 7 distinct populations of stromal cells including mesenchymal stromal cells (MSCs) ( Lepr, Adipoq, and Cxcl12), osteo-lineage cells (OLCs) ( Bglap, Spp1, and Sp7), fibroblasts ( S100a4, Fn1, and Dcn), chondrocytes ( Col2a1, Sox9, and Acan), pericytes ( Acta2, Myh11, and Mcam), and two endothelial cell (ECs) populations, arterial (AEC) and sinusoidal (SEC) ( Cdh5, Cd34, and Pecam1). Compared to normal BM, MM engraftment was associated with numerical differences in stromal cell populations between normal and MM. Gene set enrichment analysis showed an inflammatory and oxidative stress signal associated with the MM microenvironment. Sub-clustering analysis showed MSC differentiation was polarized away from osteocyte formation towards adipocytes with the identification of a novel population only seen in MM. Bone marrow endothelial cell populations were also substantially altered at this early disease stage, with differentiation polarized towards sinusoidal endothelial cells generating a pro-angiogenic /pro-inflammatory phenotype. An increase in cells undergoing endothelial to mesenchymal transition (EndMT) was also present. Taken together, we show, for the first time, existence of remodeling of the stromal populations induced by the MM clone characterized by a pro-inflammatory phenotype together with polarized differentiation. These changes result in the expansion of a number of key populations that increase the MM niche contributing to growth and survival signals and shaping of the content of the immune microenvironment. These changes result in a self-perpetuating signaling loop between cells, which needs to be broken therapeutically in order to stop progression, induce remission and long-term disease outcomes. Conclusion - In early stages of MM pathogenesis, MM cells remodel the stromal microenvironment by altering the amount and function of MSCs and endothelial cells. Through favoring an adipocytic fate of MSCs, endothelial mesenchymal transition and altering the balance between arterial and sinusoidal endothelial cells, MM cells promote an inflammatory environment that contributes to MM development and progression.