Stromal Reaction Research Articles

Cancer-Associated Fibroblasts in Pancreatic Ductal Adenocarcinoma or a Metaphor for Heterogeneity: From Single-Cell Analysis to Whole-Body Imaging.

Pancreatic ductal adenocarcinoma (PDAC) represents a formidable challenge due to its aggressive nature and poor prognosis. The tumor microenvironment (TME) in PDAC, characterized by intense stromal desmoplastic reactions and a dominant presence of cancer-associated fibroblasts (CAFs), significantly contributes to therapeutic resistance. However, within the heterogeneous CAF population, fibroblast activation protein (FAP) emerges as a promising target for Gallium-68 FAP inhibitor positron emission tomography (Ga68FAPI-PET) imaging. Notably, 68Ga-FAPI-PET demonstrates promising diagnostic sensitivity and specificity, especially in conjunction with low tracer uptake in non-tumoral tissues. Moreover, it provides valuable insights into tumor-stroma interactions, a critical aspect of PDAC tumorigenesis not adequately visualized through conventional methods. The clinical implications of this innovative imaging modality extend to its potential to reshape treatment strategies by offering a deeper understanding of the dynamic TME. However, while the potential of 68Ga-FAPI-PET is evident, ongoing correlative studies are essential to elucidate the full spectrum of CAF heterogeneity and to validate its impact on PDAC management. This article provides a comprehensive review of CAF heterogeneity in PDAC and explores the potential impact of 68Ga-FAPI-PET on disease management.

Quantitative morphometry of compensated liver cirrhosis can aid to predict the outcomes of surgical treatment

Objective — to define quantitative morphometric characteristics of hepatic parenchyma lesions at compensated liver cirrhosis (CLC), which will allow more accurately to assess its functional reserves and predict the outcomes of surgical treatment. Materials and methods. Intraoperative marginal liver biopsy was performed for 74 patients with CLC, who underwent surgical treatment. Morphological examinations with quantitative morphometry of intraoperative liver biopsies were done with the use of cytological analyzer with software «Integral‑2MT» produced by the association «Kvant» (Kyiv). The connective tissue area (CTA), unchanged hepatocytes area (UHA), stromal‑parenchymatous ratio (SPR), the volume of hepatocytes in the state of necrosis and/or necrobiosis (VHSNN), the volume of hepatocytes in the state of division (VHSD) were determined. The statistical analysis was performed by use of Statistica 12 software package. Results. Three types of morphological pattern of CLC were isolated. Type I (type A) portal cirrhosis with weakly pronounced signs of parenchymatous and stromal reaction (mono‑multilobular type, portal cirrhosis). The CTA was 66.73±1.71 mm2, SPR was 0.285±0.019, UHA was 234.13±11.5 mm2, VHSNN was 11.21±0.74%, VHSD was 10.23±0.57%. Type II cirrhosis (type B) — cirrhosis of mono‑multilobular type with moderately expressed signs of parenchymatous and stromal reaction. The CTA was 126.69±12.5 mm2, SPR was 0.617±0.031, UHA was 205.34±13.8 mm2, VHSNN was 17.32±0.63%, VHSD was 15.43±0.48%. Type III cirrhosis (type C) — cirrhosis with pronounced signs of parenchymatous and stromal reaction, more often of multilobular type. The CTA was 240.16±13.4 mm2, SPR was 1.344±0.089, UHA was 178.69±18.7 mm2, VHSNN was 23.97±0.75%, VHSD was 11.07±0.58%. Analysis of immediate results of surgical treatment demonstrated, that no one patient with A‑type CLC died, while 9.5% of patients with B‑type and 25.0% of patients with C‑type CLC died in the early postoperative period. Conclusions. Quantitative morphometry demonstrated that pathological changes in the liver at CLC are heterogeneous and can be categorized in to 3 types (A, B, C). Three types of morphological pattern at CLC, which were isolated, are characterized by significant differences in CTA, UHA, SPR, VHSNN, VHSD parameters. At transition of A‑type cirrhosis into C‑type, volume of hepatic parenchyma becomes to be decreased, while volume of connective tissue becomes to be increased. This is accompanied by decrease in UHA, increase in CTA, SPR and VHSNN. Surgical intervention in patients with type C morphological pattern of cirrhosis is associated with a high risk (25%) of poor outcome, which indicates the need to limitations for indications to elective surgical operations in this category of patients. Thus, quantitative morphometry of liver biopsies in CLC patients can aid to predict the outcomes of surgical treatment.

Deep-learning model to improve histological grading and predict upstaging of atypical ductal hyperplasia / ductal carcinoma in situ on breast biopsy.

Risk stratification of atypical ductal hyperplasia (ADH) and ductal carcinoma in situ (DCIS), diagnosed using breast biopsy, has great clinical significance. Clinical trials are currently exploring the possibility of active surveillance for low-risk lesions, whereas axillary lymph node staging may be considered during surgical planning for high-risk lesions. We aimed to develop a machine-learning algorithm based on whole-slide images of breast biopsy specimens and clinical information to predict the risk of upstaging to invasive breast cancer after wide excision. Patients diagnosed with ADH/DCIS on breast biopsy were included in this study, comprising 592 (740 slides) and 141 (198 slides) patients in the development and independent testing cohorts, respectively. Histological grading of the lesions was independently evaluated by two pathologists. Clinical information, including biopsy method, lesion size, and Breast Imaging Reporting and Data System (BI-RADS) classification of ultrasound and mammograms, were collected. Deep DCIS consisted of three deep neural networks to evaluate nuclear grade, necrosis, and stromal reactivity. Deep DCIS output comprised five parameters: total patches, lesion extent, Deep Grade, Deep Necrosis, and Deep Stroma. Deep DCIS highly correlated with the pathologists' evaluations of both slide- and patient-level labels. All five parameters of Deep DCIS were significantly associated with upstaging to invasive carcinoma in subsequent wide excisional specimens. Using multivariate logistic regression, Deep DCIS predicted upstaging to invasive carcinoma with an area under the curve (AUC) of 0.81, outperforming pathologists' evaluation (AUC, 0.71 and 0.69). After including clinical and hormone receptor status information, performance further improved (AUC, 0.87). This combined model retained its predictive power in two subgroup analyses: the first subgroup included unequivocal DCIS (excluding cases of ADH and DCIS suspicious for microinvasion) (AUC, 0.83), while the second excluded cases of high-grade DCIS (AUC, 0.81). The model was validated in an independent testing cohort (AUC, 0.81). This study demonstrated that deep-learning models can refine histological evaluation of ADH and DCIS on breast biopsies, which may help guide future treatment planning.

Proportion and distribution of PD-L1 expression and associated clinicopathologic features in patients with microsatellite instability-high gastric cancer.

387 Background: The frequency of microsatellite instability-high (MSI-H) phenotype in sporadic gastric cancers (GCs) widely varies from 8% to 20%. From prior data, MSI-H phenotypes are significantly associated with female sex, older age, antral location of the tumor, well to moderate differentiation, intestinal type, non-signet ring cell component, mucinous histologic type, a moderate to severe lymphoid stromal reaction, and a lower TNM stage. This study investigated the proportion and distribution of PD-L1 expression of MSI-H GCs compared to microsatellite stable (MSS) GCs. Methods:The study consists of 847 cases of unselected GC. MSI analysis was performed on all GC cases by immunohistochemistry for mismatch repair proteins followed by multiplex polymerase chain reaction. Among them, 72 GCs were found to be an MSI-H phenotype. After matching the T category of GCs, 200 cases of GC with MSS phenotype were selected for a control group. Immunohistochemistry for PD-L1 (SP263) was performed on 72 MSI-H GCs and 200 MSS GCs. Results: Patients with MSI-GC were significantly associated with advanced age (p <0.001), advanced tumor (p=0.0173), antral location of the tumor(p=0.0018), and T2 and T3 category (p=0.0031) but not T1 or T4 category. Histologically, MSI-H GCs occurred frequently in ulcerofungating mass (45.8%, p < 0.0001)), intestinal type (73.6%, p=0.0102), well to moderately differentiated tumor (56.9%, p=0.0102), tubular histologic type (66.7%, p=0.0013), tumor necrosis (18.1%, p <0.0001), and peritumoral neutrophilic infiltrate (40.3%, p <0.001) compared to those of 200 MSS GCs. In the evaluation of PD-L1, 55 (76.4%) MSI-H tumors revealed PD-L1 expression with ≥ 1 CPS. The predominant PD-L1 staining was found in intratumoral and peritumoral immune cells (66.7%) than within tumor cells (9.7%). The distribution of positive immune cells was predominantly located in the periphery of the tumor (37.5%) than within the tumor (9.7%). According to the criteria for PD-L1 positivity as at least 10 CPS, approximately 50% of MSI-H GCs were considered PD-L1 positive tumors with at least 10 CPS. Conclusions: In this study, the prevalence of MSI-H phenotype is approximately 8.5% of unselected GCs. Approximately 76% of patients with MSI-H tumors showed PD-L1 expression with at least 1 CPS, and 50% revealed PD-L1 expression with at least 10 CPS, who can respond better to immune checkpoint inhibitors with promising therapeutic benefits. Due to the peritumoral expression of immune cells in PD-L1 positive GCs with MSI-H phenotype, different endoscopic approaches to obtain tumor tissues for PD-L1 evaluation would be considered, such as sampling tissues at the tumor periphery.

Abstract A052: Pharmacologic conversion of cancer-associated fibroblasts from a protumor phenotype to an antitumor phenotype improves the sensitivity of pancreatic cancer to immune checkpoint blockade therapy

Abstract Background/Purpose: Cancer-associated fibroblasts (CAFs) are one of the major cell subsets in the tumor microenvironment. Their proliferation is observed in almost all invasive cancers but is most prominent in aggressive cancers with marked desmoplastic stromal reactions, such as pancreatic ductal adenocarcinoma (PDAC). Recent single-cell transcriptomic analyses have revealed CAF heterogeneity. One approach to classify CAFs is based on their cellular roles in cancer progression: cancer-promoting CAFs (pCAFs) and cancer-restraining CAFs (rCAFs). We previously identified the glycosylphosphatidylinositol-anchored protein Meflin as a rCAF-specific marker and showed that Meflin-positive CAFs are converted into Meflin-negative CAFs during cancer progression by cell lineage experiments. Interestingly, another previous study demonstrated that the number of Meflin+ rCAFs positively correlated with the response to immune checkpoint blockade (ICB) therapy in patients with non-small cell lung cancer (NSCLC). This was also shown in mouse models, where tumors developed in Meflin KO mice and mice that overexpress Meflin showed lower and higher sensitivity to anti-PD1 antibody treatment, respectively. These data suggest the significance of the balance between pCAFs and rCAFs in ICB therapy, being consistent with recent studies that have shown the critical role of CAFs in anti-tumor immunity So, The aim of this study is to establish the effects of strategies to increase the number of rCAFs on the sensitivity to ICB therapy in PDAC. Material/Methods: A search for chemical compounds that upregulate Meflin expression was performed. We then investigated the effect of the compound on the efficacy of anti-PD-L1 antibody treatment using mouse murine models of PDAC. We also addressed the mechanism by which Meflin+ rCAFs shape the immune tumor microenvironment that potentiates the efficacy of ICB therapy. Results: We performed a chemical library screen for compounds that convert protumor CAFs into rCAFs by inducing Meflin expression. As a result, they identified the synthetic retinoid AM80 as a candidate drug that achieves that CAF conversion. Next, we observed that the induction of a Meflin-positive CAF subset with a cancer-suppressive role through the oral administration of Am80 improves intratumoral delivery and efficacy of anti-PD-L1 antibody treatment in pancreatic cancer models. Interestingly, Am80 exerted this effect when administered to tumors prior to, but not concomitant with, anti-PD-L1 antibody in wild-type but not Meflin-deficient mice. Meflin+ rCAFs induced polarization of macrophages to the M1-like phenotype and durable anti-PD-L1 treatment-mediated anti-tumor responses. Conclusion: Our data suggest that strategy of pharmacologic conversion of CAF from protumor phenotype to an antitumor phenotype CAF can improves the sensitivity of pancreatic cancer to ICB therapy. Citation Format: Tadashi Iida, Owaki Takayuki, Kota Uetsuki, Yamao Kentaro, Yasuyuki Mizutani, Takuya Ishikawa, Atsushi Enomoto, Hiroki Kawashima. Pharmacologic conversion of cancer-associated fibroblasts from a protumor phenotype to an antitumor phenotype improves the sensitivity of pancreatic cancer to immune checkpoint blockade therapy [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 A052.

Histological parameters and stromal desmoplastic status affecting accurate diagnosis of extraprostatic extension of prostate cancer using multi-parametric magnetic resonance imaging.

To investigate the clinicopathological factors affecting discrepancies between multi-parametric magnetic resonance imaging (mpMRI) and histopathological evaluation for diagnosis of extraprostatic extension (EPE) of prostate cancer. One hundred-and-three lesions from 96 cases with suspected EPE on preoperative mpMRI, of which 60 and 43 showed bulging and frank capsular breach, respectively, were grouped according to pathological (p)EPE in radical prostatectomy specimens. Additionally, clinicopathological/immunohistochemical findings for periostin reflecting a desmoplastic stromal reaction were compared between these groups. pEPE was detected in 49 (48%) of the 103 lesions. Of these, 25 (42%) showed bulging and 24 (56%) showed frank capsular breach on MRI. In the total cohort, the absence of pEPE was significantly associated with a lower Gleason Grade Group (GG) (p < 0.0001), anterior location (p = 0.003), absence of intraductal carcinoma of the prostate (IDC-P) (p = 0.026), and high stromal periostin expression (p < 0.0001). These trends were preserved in subgroups defined by MRI findings, except for anterior location/IDC-P in the bulging subgroup. GG, anterior location, and periostin expression may cause mpMRI-pathological discrepancies regarding EPE. Periostin expression was a significant pEPE-negative factor in all subgroup analyses. Our results indicate that patients with suspected EPE on MRI, regardless of their pEPE results, should be followed as carefully as those with definite pEPE.

Age-Related Decline in Pancreas Regeneration Is Associated With an Increased Proinflammatory Response to Injury

Background and AimsThe regenerative capacity of the pancreas diminishes with age. Understanding acinar cell responses to injury and the resolution of regenerative processes is crucial for tissue homeostasis. However, knowledge about the impact of aging on these processes remains limited. MethodsTo investigate the influence of aging on pancreas regeneration, we established a cohort of young (7-14 weeks) and old (18 months) C57bl/6 mice. Experimental pancreatitis was induced using caerulein, and pancreas samples were collected at various timepoints after induction, covering acute damage response, inflammation, peak proliferation, and inflammation resolution. Our analysis involved immunohistochemistry, quantitative imaging, and gene expression analyses.Our study revealed a significant decline in the regenerative capacity of the pancreas in old mice. Despite similar morphology and transcriptional profiles between the pancreas of young and old mice under homeostasis, the aged pancreas is primed to generate an exacerbated proinflammatory reaction in response to injury. Specifically, we observed notable upregulation of Junb expression in acinar cells and aberrant myofibroblast activation in aged pancreas. ConclusionsThe response of acinar cells to injury in the pancreas of aged mice is characterized by an increased susceptibility to inflammation and stromal reactions. Our findings uncover a pre-existing proinflammatory state in aged acinar cells, offering insights into potential strategies to prevent the onset of pancreatic insufficiency and the development of inflammatory conditions. These insights hold implications for preventing conditions such as chronic pancreatitis and pancreatic ductal adenocarcinoma.

Histologic, immunohistochemical, and in situ hybridization study of myxoid stroma in feline oral squamous cell carcinoma.

Oral squamous cell carcinoma (oSCC) is a highly invasive malignant neoplasm in cats. Recently, tumor stroma, known as tumor microenvironments, have been considered to play an essential role in tumor progression. However, their role in feline squamous cell carcinoma (SCC) remains unclear. This study aimed to reveal the cancer microenvironment of feline oSCC and evaluate the pathological mechanisms of progression. We used 19 samples from 17 cats with oSCC, which were examined using light microscopy, immunohistochemistry, and in situ hybridization (RNAscope®). Feline oSCCs had two types of stroma, namely fibrotic and myxoid stromal reaction patterns, which were easily distinguished using hematoxylin-eosin staining. The myxoid stroma was rich in hyaluronic acid, which seems to be produced by neoplastic cells. Furthermore, the presence of myxoid stroma was correlated with histological parameters, including the appearance of cancer-associated fibroblasts and tumor budding. Periostin protein expression was also frequently observed in the stroma of feline oSCC and was significantly more common in the myxoid stromal reaction pattern group than in the fibrotic group. Positive signals for periostin mRNA were detected in stromal cancer-associated fibroblasts. This study indicates that the interaction between neoplastic cells and stromal reaction pattern components, such as hyaluronic acid and periostin, may be involved in tumor malignancy. Therefore, we propose that focus be placed not only on the tumor tissue but also on the characterization of the stroma for analyzing feline oSCC.

Investigation of the Relationship Between Tumor Microenvironment and Prognostic Parameters in Invasive Breast Carcinomas of No Special Type: A Retrospective Analysis.

The tumor microenvironment is a heterogeneous and constantly changing territory that plays an active role in tumor formation and progression. It constantly interacts with tumor cells, plays an active role in tumor development, and even appears as a parameter of prognostic importance, and the importance of the tumor microenvironment in breast cancer has been emphasized by recent studies. In this study, we aimed to retrospectively evaluate the relationship between the tumor microenvironment and prognostic parameters in invasive breast carcinomas of no special type. A total of 271 cases diagnosed as invasive breast carcinoma of no special type from resection materials in our center between 2007 and 2015 were included in the study. Hematoxylin-eosin stained slides with a thickness of 4-5 micrometers were evaluated in terms of tumor infiltrating lymphocytes, peritumoral and intratumoral desmoplastic reaction, intratumoral and peritumoral tumor budding, stromal features, and tumor growth pattern. When parameters related to the tumor microenvironment were compared with other prognostic parameters, there was a significant relationship between TILs and tumor grade, size, stage, immunohistochemical subgroup and Ki-67 proliferation index. A significant relationship was detected between intratumoral stromal reaction and tumor grade, size, molecular subgroup and the Ki-67 proliferation index (p < 0.05). When stroma and other prognostic parameters were compared, tumors with desmoplastic stroma had higher grades and higher Ki-67 proliferation indexes, and they were observed more frequently in the triple negative molecular subgroup. We believe that including parameters related to tumor microenvironment in breast cancer reports, which hold a prognostic and predictive importance, will contribute to patient management. Considering the fact that these can be easily evaluated from routinely used hematoxylin-eosin stained slides, this does not cause additional costs or excessive time loss.

Stroma AReactive Invasion Front Areas (SARIFA) improves prognostic risk stratification of perioperative chemotherapy treated oesophagogastric cancer patients from the MAGIC and the ST03 trial.

Tumour-associated fat cells without desmoplastic stroma reaction at the invasion front (Stroma AReactive Invasion Front Areas (SARIFA)) is a prognostic biomarker in gastric and colon cancer. The clinical utility of theSARIFA status in oesophagogastric cancer patients treated with perioperative chemotherapy is currently unknown. TheSARIFA status was determined in tissue sections from patients recruited into the MAGIC (n = 292) or ST03 (n = 693) trials treated with surgery alone (S, MAGIC) or perioperative chemotherapy (MAGIC, ST03). The relationship between SARIFA status, clinicopathological factors, overall survival (OS) and treatment was analysed. TheSARIFA status was positive in 42% MAGIC trial S patients, 28% MAGIC and 48% ST03 patients after pre-operative chemotherapy. SARIFA status was related to OS in MAGIC trial S patients and was an independent prognostic biomarker in ST03 trial patients (HR 1.974, 95% CI 1.555-2.507, p < 0.001). ST03 patients with lymph node metastasis (ypN + ) and SARIFA-positive tumours had poorer OS than patients with ypN+ and SARIFA-negative tumours (plogrank < 0.001). TheSARIFA status has clinical utility as prognostic biomarker in oesophagogastric cancer patients irrespective of treatment modality. Whilst underlying biological mechanisms warrant further investigation, theSARIFA status might be used to identify new drug targets, potentially enabling repurposing of existing drugs targeting lipid metabolism.

Abstract IA008: Mechanisms and consequences of pancreatic cancer stromal evolution

Abstract The mechanisms underlying evolution of tumor-associated stroma remain poorly understood. In solid tumors featuring a prominent stromal reaction, an improved understanding of the functions and origins of abundant stromal cell types may facilitate the development of new and effective therapies. Pancreatic ductal adenocarcinoma (PDAC) is the quintessence of a fibro-inflammatory malignancy, with 50-90% of tumor volume occupied by a dense, desmoplastic stroma. Cancer-associated fibroblasts (CAFs) are the key cell type which drives the stromal reaction in PDAC, and recent reports suggest that stromal CAFs represent a heterogeneous population of cells from diverse origins, potentially including cell types which support and others which suppress tumor growth. Pancreatic stellate cells (PSCs) are lipid-storing cells in healthy pancreas which can transdifferentiate to an activated CAF phenotype. PSCs have been suggested as the predominant source of fibroblasts in the PDAC tumor microenvironment. However, proper lineage tracing studies have never been performed, such that the relative contribution and specific functions of PSCs in the tumor microenvironment are unknown. We have developed a novel mouse model to track PSC differentiation and function during pancreatic tumor progression in vivo. This model revealed that PSC-derived CAFs in fact give rise to a numerically minor but functionally significant subset of PDAC CAFs, and may represent a viable therapeutic target. We adapted our mouse model to enable targeted ablation of PSCs and derivative CAFs within their host tissue for the first time. Functional studies in this model reveal non-redundant functions for PSC-derived CAFs in shaping the PDAC microenvironment, and highlight the significance of mesenchymal lineage heterogeneity for pancreatic tumorigenesis. Further, we find that tumor genotype with respect to p53 status dictates stromal evolutionary routes with respect to cell of origin, a finding that may extend to additional cancer cell-intrinsic genomic alterations. Together, these findings shed light on mechanisms that shape the tumor microenvironment during pancreatic cancer progression, and may hold relevance in additional solid tumor types. Citation Format: Mara Sherman. Mechanisms and consequences of pancreatic cancer stromal evolution [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 IA008.

Stiffness-induced cancer-associated fibroblasts are responsible for immunosuppression in a platelet-derived growth factor ligand-dependent manner.

Pancreatic ductal adenocarcinoma (PDAC) is associated with a vast stromal reaction that arises mainly from cancer-associated fibroblasts (CAFs) and promotes both immune escape and tumor growth. Here, we used a mouse model with deletion of the activin A receptor ALK4 in the context of the KrasG12D mutation, which strongly drives collagen deposition that leads to tissue stiffness. By ligand-receptor analysis of single-cell RNA-sequencing data, we identified that, in stiff conditions, neoplastic ductal cells instructed CAFs through sustained platelet-derived growth factor (PDGF) signaling. Tumor-associated tissue rigidity resulted in the emergence of stiffness-induced CAFs (siCAFs) in vitro and in vivo. Similar results were confirmed in human data. siCAFs were able to strongly inhibit CD8+ T-cell responses in vitro and in vivo, promoting local immunosuppression. More importantly, targeting PDGF signaling led to diminished siCAF and reduced tumor growth. Our data show for the first time that early paracrine signaling leads to profound changes in tissue mechanics, impacting immune responses and tumor progression. Our study highlights that PDGF ligand neutralization can normalize the tissue architecture independent of the genetic background, indicating that finely tuned stromal therapy may open new therapeutic avenues in pancreatic cancer.

Abstract A013: Targeting perivascular macrophages with an orally bioavailable heme oxygenase-1 inhibitor improves responses to chemotherapeutic drugs in cancer

Abstract The tumor microenvironment (TME) plays a crucial role in cancer progression. It consists of various elements including stromal cells, proteins, and physical factors that support the growth and spread of cancer. Targeting the TME has become an appealing strategy for cancer treatment. Tumor-associated macrophages (TAMs) are an abundant stromal cell type in the TME and, due to their plasticity, TAMs can be exploited by cancer cells and the broader stromal reaction to promote a pro-tumoral response. TAMs have been implicated in facilitating a variety of pro-tumoral pathways including resistance to anti-cancer therapeutics, immune suppression, angiogenesis, and metastasis. Our group has recently characterized a subpopulation of pro-angiogenic and immune-suppressive TAMs which can be identified by their expression of the lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1). LYVE-1+ TAMs reside spatially proximal to blood vasculature and adopt a collaborative multi-cellular ‘nest’ arrangement which may support their biological activity. LYVE-1+ TAMs selectively express high levels of the immunomodulatory enzyme heme oxygenase-1 (HO-1), an enzyme which catabolizes heme to generate the biologically active catabolites carbon monoxide (CO), biliverdin and ferrous iron. Genetic inactivation of HO-1 in LYVE-1+ TAMs results in improved anti-tumoral CD8 T-cell responses and enhanced response to chemotherapy, resulting in prolonged tumor control in a spontaneous MMTV-PyMT murine model of breast cancer. Unfortunately, current HO-1 inhibitors are not orally bioavailable, which limits their utility as immunotherapeutics for the treatment of cancer. In response to this hurdle, we have developed a next generation HO-1 inhibitor called KCL-HO-1i. We demonstrate in pharmacokinetic studies that KCL-HO-1i is orally bioavailable in murine models with a serum half-life of around 3hours. Furthermore, orally delivering KCL-HO-1i alongside standard of care chemotherapy is able to deliver durable tumor control in MMTV-PyMT mice. We have characterized the response of the TME to KCL-HO-1i using flow cytometry, immunofluorescence and RNA sequencing approaches and demonstrate that KCL-HO-1i supports a broader switch from an immunological ‘cold’ to ‘hot’ TME which provides a more favourable immune landscape for improving the response to chemotherapeutic drugs. Taken together, our data support the use of KCL-HO-1i as novel immunotherapeutic for the treatment of cancer. Citation Format: Meriem Bahri, Taha Al-Adhami, Emre Demirel, Joanne E. Anstee, Karen T. Feehan, James Rosekilly, Cheryl E. Gillett, Dominika Sosnowska, Renee Gitsaki-Taylor, Tik Shing Cheung, James F. Spicer, Khondaker Miraz Rahman, James N. Arnold. Targeting perivascular macrophages with an orally bioavailable heme oxygenase-1 inhibitor improves responses to chemotherapeutic drugs in cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr A013.

Glutamine addiction and therapeutic strategies in pancreatic cancer

Pancreatic cancer remains one of the most lethal diseases worldwide owing to its late diagnosis, early metastasis, and poor prognosis. Because current therapeutic options are limited, there is an urgent need to investigate novel targeted treatment strategies. Pancreatic cancer faces significant metabolic challenges, principally hypoxia and nutrient deprivation, due to specific microenvironmental constraints, including an extensive desmoplastic stromal reaction. Pancreatic cancer cells have been shown to rewire their metabolism and energy production networks to support rapid survival and proliferation. Increased glucose uptake and glycolytic pathway activity during this process have been extensively described. However, growing evidence suggests that pancreatic cancer cells are glutamine addicted. As a nitrogen source, glutamine directly (or indirectly via glutamate conversion) contributes to many anabolic processes in pancreatic cancer, including amino acids, nucleobases, and hexosamine biosynthesis. It also plays an important role in redox homeostasis, and when converted to α-ketoglutarate, glutamine serves as an energy and anaplerotic carbon source, replenishing the tricarboxylic acid cycle intermediates. The present study aims to provide a comprehensive overview of glutamine metabolic reprogramming in pancreatic cancer, focusing on potential therapeutic approaches targeting glutamine metabolism in pancreatic cancer.

MSC Reprogramming and Aberrant Inflammatory Signaling in Myelofibrosis: Insights from Single-Cell RNA-Sequencing of Trephine Bone Marrow Cells

Myelofibrosis (MF) results from mutation-induced activation of JAK/STAT signaling in hematopoietic stem, progenitor and mature cells, and features a progressive, fibrosing stromal reaction, splenomegaly, constitutional symptoms and reduced life expectancy. Identification of disrupted cellular pathways within bone marrow (BM) is pivotal for therapeutic breakthrough, but fibrosis severely limits access to stromal elements for single-cell analysis. To overcome this, we utilized collagenase digestion of fresh BM trephine biopsy material surplus to diagnostic needs and profiled isolated cells using scRNAseq. 9 samples were obtained from 8 patients with MF (4, JAK inhibitor naïve; 4, on ruxolitinib); 4 samples were from 4 patients with MF 4, 10, 12 &amp; 12 months after allogeneic transplant; and 7 samples (termed “normal controls”) were from 6 patients who had received an allograft for myelodysplasia or acute leukemia, and 1 who had received CAR-T cells for B-ALL. Normal controls exhibited normal or near-normal blood counts and lacked BM fibrosis. Each sample was subjected to collagenase digestion and flow sorted to isolate non-erythroid hematopoietic (CD45+GlyA-) and microenvironmental (CD45+GlyA-CD71-) cells for single cell RNA sequencing and downstream comparative analysis. 105,231 transcriptomes met quality control criteria (median 4975/sample, range 1036-13277, no significant difference between sample categories) and 43 transcriptional clusters were identified. Compositional analysis identified the non-hematopoietic stromal cell fraction as harboring the biggest differences with, surprisingly, a proportional relative depletion of mesenchymal stromal cells (MSCs) and enrichment of endothelial cells (ECs) in MF vs. normal controls. While transcriptomes of ECs did not differ substantially, there were extensive transcriptional differences between MSCs recovered from MF vs. normal control samples. Subclustering revealed two subsets, MSC1 &amp; MSC2 asymmetrically distributed between MF and normal controls. Intriguingly, MSCs recovered from MF patients after allograft occupied an intermediate position in principal component space indicating that reversion of disease-related MSC transcriptional reprogramming post-allograft occurs over a timescale of months. The reprogrammed transcriptional state of MF MSCs is defined by reduced expression of hematopoietic support factor genes (eg, KITLG, CXCL12, ANGPTL2, NCAM2, CSF1) and increased expression of extracellular matrix (ECM) genes (eg, FN1, COL1A2, COL3A1, DCN, SPARC), features which suggest an explanation for the increased frequency of graft failure following allograft in MF versus other hematologic malignancies. Single-Cell rEgulatory Network Inference and Clustering (SCENIC) indicated transcription factors with either increased (eg, NR2F2, RUNX2, FOXP2) or decreased (eg, CEBPA, FOXC1, and PPARG) transcriptional activity as candidate master transcriptional regulators of the observed MSC reprogramming in MF. Transcriptional change in MSCs correlated strongly with histopathological assessment of BM reticulin. Within the hematopoietic fraction, and agnostic of JAK inhibitor treatment status, comparison of MF vs. control cells unexpectedly revealed significant upregulation of interferon response genes, especially in CD14 + monocytes, but also in neutrophils and their progenitors, centred on expression of interferon-stimulated gene factor 3 (ISGF3) genes IRF9 and STAT1/2. Comparison of MF samples according to ruxolitinib treatment status indicated that ruxolitinib induced downregulation of an NFκB-centered inflammatory program, particularly in neutrophils. Finally, ligand-receptor analysis using NicheNet identified TGFB1, IL1B, and TNF as top predicted ligands for induction of the MF signature in MSCs. While myeloid cells were top producers of these factors, their expression did not vary between MF vs. controls, suggesting other mechanisms, such as prolonged retention of ligand in the ECM, are important. In summary, our comparative scRNA analysis of BM cells from MF patients and controls - the largest reported to date - reveals (i) transcriptional reprogramming of MSCs leading to reduced expression of hematopoietic support factor and increased expression of ECM genes; and (ii) increased expression of interferon and NFkB inflammatory pathways in myeloid cells.

Bronchial oncocytic carcinoma in an adult: a case report and literature review

BackgroundLung salivary-type tumors originating from bronchial submucosal glands are rare, only four types of salivary gland-type tumors are listed in 2015 WHO classification of lung tumors. Here, we report a rare case of oncocytic carcinoma (OC) in the right main bronchus.Case presentationA 34-year-old man presented to our hospital with a two-month history of recurrent hemoptysis and with one month of inspiratory dyspnea. Pulmonary function tests showed mild restrictive ventilatory dysfunction and severe diffusion dysfunction. Furthermore, the flow volume loop showed a variable extra-thoracic obstruction. Computed tomography (CT) of the chest revealed that a polypiform nodule of 13 mm in diameter was at the proximal right main bronchus. Testing for purified protein derivative was positive (category 2). The nodule was resected under bronchoscopy. The bronchial aspirate was negative for mycobacterium tuberculosis and tumor cells. The biopsy sample showed a solid and acinar predominant pattern with abundant eosinophilic cytoplasm. The bronchial mucosa was destroyed and replaced by tumor cells. The loose edematous stromal reaction could be seen in a local area. Immunohistochemically, tumor cells were positive for CK, EMA, Vimentin, CD117, CK7, S100, Mammaglobin and SOX10. Only scattered tumor cells were stained by basal cell markers, including CK5/6, P40 and P63. Electron microscopy revealed numerous swelling mitochondria with lacking mitochondrial cristae in tumor cells. Fluorescence in situ hybridization (FISH) testing for MAML2 and ETV6 rearrangement were negative. Next-generation sequencing analysis of 520 genes in the tissue biopsy specimen showed no somatic mutation. The diagnosis of OC was made. Subsequently, the patient underwent a right upper lobectomy with sleeve resection of the main bronchus and lymph dissection. No recurrent evidence was seen during two years of chest CT follow-up.ConclusionsTo our knowledge, this is the first case of primary OC in the bronchus. This patient has no recurrence during two years of follow-up, indicating that primary OC in the bronchus has the same favorable prognosis as in salivary glands. Moreover, complete excision and thorough sampling to know the invasive growth pattern is important to reach the correct diagnosis.

Meflin is a marker of pancreatic stellate cells involved in fibrosis and epithelial regeneration in the pancreas.

Pancreatic stellate cells (PSCs) are stromal cells in the pancreas that play an important role in pancreatic pathology. In chronic pancreatitis (CP) and pancreatic ductal adenocarcinoma (PDAC), PSCs are known to get activated to form myofibroblasts or cancer-associated fibroblasts (CAFs) that promote stromal fibroinflammatory reactions. However, previous studies on PSCs were mainly based on the findings obtained using ex vivo expanded PSCs, with few studies that addressed the significance of in situ tissue-resident PSCs using animal models. Their contributions to fibrotic reactions in CP and PDAC are also lesser-known. These limitations in our understanding of PSC biology have been attributed to the lack of specific molecular markers of PSCs. Herein, we established Meflin (Islr), a glycosylphosphatidylinositol-anchored membrane protein, as a PSC-specific marker in both mouse and human by using human pancreatic tissue samples and Meflin reporter mice. Meflin-positive (Meflin+ ) cells contain lipid droplets and express the conventional PSC marker Desmin in normal mouse pancreas, with some cells also positive for Gli1, the marker of pancreatic tissue-resident fibroblasts. Three-dimensional analysis of the cleared pancreas of Meflin reporter mice showed that Meflin+ PSCs have long and thin cytoplasmic protrusions, and are localised on the abluminal side of vessels in the normal pancreas. Lineage tracing experiments revealed that Meflin+ PSCs constitute one of the origins of fibroblasts and CAFs in CP and PDAC, respectively. In these diseases, Meflin+ PSC-derived fibroblasts showed a distinctive morphology and distribution from Meflin+ PSCs in the normal pancreas. Furthermore, we showed that the genetic depletion of Meflin+ PSCs accelerated fibrosis and attenuated epithelial regeneration and stromal R-spondin 3 expression, thereby implying that Meflin+ PSCs and their lineage cells may support tissue recovery and Wnt/R-spondin signalling after pancreatic injury and PDAC development. Together, these data indicate that Meflin may be a marker specific to tissue-resident PSCs and useful for studying their biology in both health and disease. © 2023 The Pathological Society of Great Britain and Ireland.

Cancer-Associated Fibroblasts Influence Survival in Pleural Mesothelioma: Digital Gene Expression Analysis and Supervised Machine Learning Model.

The exact mechanism of desmoplastic stromal reaction (DSR) formation is still unclear. The interaction between cancer cells and cancer-associated fibroblasts (CAFs) has an important role in tumor progression, while stromal changes are a poor prognostic factor in pleural mesothelioma (PM). We aimed to assess the impact of CAFs paracrine signaling within the tumor microenvironment and the DSR presence on survival, in a cohort of 77 PM patients. DSR formation was evaluated morphologically and by immunohistochemistry for Fibroblast activation protein alpha (FAP). Digital gene expression was analyzed using a custom-designed CodeSet (NanoString). Decision-tree-based analysis using the "conditional inference tree" (CIT) machine learning algorithm was performed on the obtained results. A significant association between FAP gene expression levels and the appearance of DSR was found (p = 0.025). DSR-high samples demonstrated a statistically significant prolonged median survival time. The elevated expression of MYT1, KDR, PIK3R1, PIK3R4, and SOS1 was associated with shortened OS, whereas the upregulation of VEGFC, FAP, and CDK4 was associated with prolonged OS. CIT revealed a three-tier system based on FAP, NF1, and RPTOR expressions. We could outline the prognostic value of CAFs-induced PI3K signaling pathway activation together with FAP-dependent CDK4 mediated cell cycle progression in PM, where prognostic and predictive biomarkers are urgently needed to introduce new therapeutic strategies.

Multifaced roles of desmoplastic reaction and fibrosis in pancreatic cancer progression: Current understanding and future directions.

Desmoplastic reaction is a fibrosis reaction that is characterized by a large amount of dense extracellular matrix (ECM) and dense fibrous stroma. Fibrotic stroma around the tumor has several different components, including myofibroblasts, collagen, and other ECM molecules. This stromal reaction is a natural response to the tissue injury process, and fibrosis formation is a key factor in pancreatic cancer development. The fibrotic stroma of pancreatic cancer is associated with tumor progression, metastasis, and poor prognosis. Reportedly, multiple processes are involved in fibrosis, which is largely associated with the upregulation of various cytokines, chemokines, matrix metalloproteinases, and other growth factors that promote tumor growth and metastasis. Fibrosis is also associated with immunosuppressive cell recruitment, such as regulatory T cells (Tregs) with suppressing function to antitumor immunity. Further, dense fibrosis restricts the flow of nutrients and oxygen to the tumor cells, which can contribute to drug resistance. Furthermore, the dense collagen matrix can act as a physical barrier to block the entry of drugs into the tumor, thereby further contributing to drug resistance. Thus, understanding the mechanism of desmoplastic reaction and fibrosis in pancreatic cancer will open an avenue to innovative medicine and improve the prognosis of patients suffering from this disease.

Leaf chlorophyll fluorescence and reflectance of oakleaf lettuce exposed to metal and metal(oid) oxide nanoparticles

BackgroundMost nanoparticles (NPs) have a significant impact on the structure and function of the plant photosynthetic apparatus. However, their spectrum of action varies significantly, from beneficial stimulation to toxicity, depending on the type of NPs, the concentration used and plant genotypic diversity. Photosynthetic performance can be assessed through chlorophyll a fluorescence (ChlF) measurements. These data allow to indirectly obtain detailed information about primary light reactions, thylakoid electron transport reactions, dark enzymatic stroma reactions, slow regulatory processes, processes at the pigment level. It makes possible, together with leaf reflectance performance, to evaluate photosynthesis sensitivity to stress stimuli. ResultsWe investigated effects of different metal and metal(oid) oxide nanoparticles on photosynthesis of oakleaf lettuce seedlings by monitoring the chlorophyll a fluorescence light radiation and reflectance from the leaves. Observations of ChlF parameters and changes in leaf morphology were carried out for 9 days in two-day intervals. Spectrophotometric studies were performed at 9th day. Suspensions of NPs with the following concentrations were used: 6% TiO2, SiO2; 3% CeO2, SnO2, Fe2O3; 0.004% (40 ppm) Ag; 0.002% (20 ppm) Au. Nanoparticles were applied directly on the leaves which caused small symptoms of chlorosis, necrosis and leaf veins deformation, but the plants fully recovered to the initial morphological state at 9th day. Leaf reflectance analysis showed an increase in FRI for SiO2-NPs and CeO2-NPs treatments and ARI2 for Fe2O3, however, WBI and PRI coefficients for the latter nanoparticle were lower than in control. Chlorophyll a fluorescence parameters have changed due to NPs treatment. Fe2O3-NPs caused an increase in Fv/F0, PIABS, ET0/RC, DI0/RC, ABS/RC in different time points in comparison to control, also Ag, Au and SnO2 treatment caused an increase in Fv/F0, PIABS or ET0/RC, respectively. On the other hand, TiO2-NPs caused a decrease in Fv/Fm and Fv/F0 parameters, but an increase in DI0/RC value was observed. SnO2-NPs decreased PIABS, but increased ET0/RC than compared to control. Nanoparticles affected the shape of the O-J-I-P curve in slight manner, however, further analyses showed unfavourable changes within the PSII antenna, manifested by a slowdown in the transport of electrons between the Chl molecules of the light-harvesting complex II and the active center of PSII due to NPs application.ConclusionChanges in ChlF parameters and leaf reflectance values clearly proved the significant influence of NPs on the functioning of the photosynthetic apparatus, especially right after NPs application. The nature of these changes was strictly depended on the type of nanoparticles and sometimes underwent very significant changes over time. The greatest changes in ChlF parameters were caused by Fe2O3 nanoparticles, followed by TiO2-NPs. After slight response of O-J-I-P curves to treatment of the plants with NPs the course of the light phase of photosynthesis stabilized and at 9th day were comparable to the control curve.