Manuela Gariboldi

Background/Objectives: OrganoHIPEC is a phase-II, two-stage, open-label clinical trial that investigates if cytoreductive surgery (CRS) and patient-tailored HIPEC, based on a preclinical platform using patient-derived organoids, can improve disease control in peritoneal metastases from colorectal cancer (CRC-PM). Methods: Adults with limited CRC-PM and no distant metastases were included. CRC-PM were sampled for organoid development during diagnostic laparoscopy. These organoids were used in an in vitro HIPEC model to test various drugs suitable for intraperitoneal administration. After 3–6 months of systemic chemotherapy, patients without progression underwent CRS/HIPEC with personalized regimens based on organoid drug response. To detect an increase in 12-month peritoneal disease-free survival from 40% to 60%, 24 patients are needed. According to the two-stage design, if n = 14), cisplatin/mitomycin-C (n = 12), and low-dose (120 min) oxaliplatin (n = 4). No patient was sensitive to high-dose oxaliplatin (30 min) and cisplatin/doxorubicin. Ten patients had a potential follow-up >12 months. Peritoneal relapse occurred at 8 months in two patients, and one died of liver metastases at 7 months. Seven patients remained PM-free for >12 months (median 16.4, range 12.6–28.4). Conclusions: A comprehensive precision approach using patient-derived organoids to guide personalized HIPEC is feasible and shows promising early results. High-dose oxaliplatin is poorly active. As 7/10 patients achieved the endpoint of 12-month PM-free survival, Stage-1 was successfully completed. The trial is proceeding to Stage-2.

Patient-derived organoids (PDOs) are tridimensional cultures derived from the stem component of a tissue. They preserve the genetic and phenotypic characteristics of the tissue of origin, and represent valuable in vitro models for drug screening, biomarker discovery, cell therapy and genetic modification. Importantly, PDOs reproduce the tumor behavior and can predict therapeutic responses, making them relevant for clinical applications for personalized therapies. PDOs may also be used for studying the interactions between cancer cells and the tumor microenvironment (TME). These interactions are driven by biochemical factors released by the cells, and biomechanical events such as the remodeling of the extracellular matrix (ECM). In recent years, it has become evident that the interactions between cancer cells and the TME have an impact on tumor development and on the efficacy of cancer therapy Therefore, targeting both tumor cells and the TME may improve patient response to treatment. Most PDO culture protocols are limited to epithelial cells. However, recent advances such as use of decellularized ECM (dECM) scaffolds have allowed for the development of in vivo-like environments that host diverse cell types, both normal and pathological, in a tridimensional (3D) manner that closely mimics the complexity of the TME. dECM-based models effectively replicate the interactions between tumor cells, ECM and the microenvironment, are easy to analyze and adaptable for drug testing. By incorporating TME components and therapeutic agents, these models offer an advanced platform for preclinical testing.

Approximately 10%–15% of all lung cancers arise in non‐smokers. Although there are no established aetiological factors, non‐smokers with a family history of cancer have an increased risk of lung cancer, implying host genetic factors in lung cancer susceptibility. We sought to identify, in a cohort of 75 patients recruited before lung lobectomy, germline alterations with a strong association with lung cancer. Whole‐exome sequencing was performed on genomic DNA from peripheral blood. Six resources were used to select pathogenic germline variants with strong clinical significance. In total, 33 pathogenic or likely pathogenic variants in 31 genes were identified. Of these, 13 were located in cancer‐predisposing genes (nine were lung cancer drivers), most of which were involved in DNA repair mechanisms and diseases of metabolism. Among DNA repair‐related genes, BRCA1 and BRCA2, and ATM have also been identified in other studies on non‐smokers. Our results strongly support the hypothesis that a number of non‐smoker lung cancer patients carry germline variants in cancer‐predisposing genes, suggesting that lung cancer patients, particularly non‐smokers, should be considered for germline molecular testing.

Introduction: Individuals with Familial Adenomatous Polyposis (FAP) or APC-associated polyposis, an autosomal dominant inherited condition, develop multiple adenomatous polyps and have an increased colorectal cancer (CRC) risk. A change in diet can help reduce cancer risk, and several dietary components have an antitumor effect. We aimed to evaluate the potential of the anti-inflammatory and anticancer substances quercetin (QER), epigallocatechin gallate (EGG) and fisetin (FIS) in decreasing the risk of CRC by reducing the growth of polyps in an organoid model. Methods: Patient-derived organoid (PDO) lines were generated from polyps obtained from patients with FAP undergoing prophylactic colectomy. PDOs were treated with QER, EGG, or FIS to determine their effect on cell growth. Changes in caspase 3/7 activity and expression of inflammation and apoptosis mediators were assessed by luminescent and colorimetric assays. Results: Three PDO lines with different inactivating pathogenic variants in the APC gene were developed using a combinatorial approach. FIS was the most active of the three substances tested, presenting the lowest IC50 in all PDO lines (range: 42.6-9.2 uM). The IC50 was defined as the concentration required to halve the number of cells after 72 hours. All molecules tested induced apoptosis through activation of caspases 3/7. Conclusions: QER, EGG, and FIS can be easily taken from foods or dietary supplements, show toxicity on PDOs derived from adenomatous polyps, while they are known to be harmless on normal cells. Diets enriched with these substances could be potential supplemental treatments to reduce the risk of CRC in individuals with FAP.

Background: Colorectal cancer (CRC) remains a significant healthcare burden worldwide, characterized by a complex interplay between obesity and chronic inflammation. While the relationship between CRC, obesity and altered lipid metabolism is not fully understood, there are evidences suggesting a link between them. In this study, we hypothesized that dysregulated lipid metabolism contributes to local accumulation of foam cells (FC) in CRC, which in turn disrupts antitumor immunosurveillance. Methods: Tumor infiltrating FC and CD8⁺ were quantified by digital pathology in patients affected by T2–T4 CRC with any N stage undergoing radical upfront surgery (n=65) and correlated with patients’ clinical outcomes. Multiparametric high-resolution flow cytometry analysis and bulk RNAseq of CRC tissue were conducted to evaluate the phenotype and transcriptomic program of immune cell infiltrate in relation to FC accumulation. The immunosuppressive effects of FC and mechanistic studies on FC-associated transforming growth factor-beta (TGF-β) and anti-PD-L1 inhibition were explored using an in-vitro human model of lipid-engulfed macrophages. Results: FC (large CD68⁺ Bodipy⁺ macrophages) accumulated at the tumor margin in CRC samples. FC^high tumors exhibited reduced CD8⁺ T cells and increased regulatory T cells (Tregs). Functional transcriptional profiling depicted an immunosuppressed milieu characterized by reduced interferon gamma, memory CD8⁺ T cells, and activated macrophages mirrored by increased T-cell exhaustion and Treg enrichment. Furthermore, FC^high tumor phenotype was independent of standard clinical factors but correlated with high body mass index (BMI) and plasma saturated fatty acid levels. In CD8^low tumors, the FC^high phenotype was associated with a 3-year disease-free survival rate of 8.6% compared with 28.7% of FC^low (p=0.001). In-vitro studies demonstrated that FC significantly impact on CD8 proliferation in TFG-β dependent manner, while inhibition of TGF-β FC-related factors restored antitumor immunity. Conclusions: FC exert immunosuppressive activity through a TGF-β-related pathway, resulting in a CD8-excluded microenvironment and identifying immunosuppressed tumors with worse prognosis in patients with primary CRC. FC association with patient BMI and dyslipidemia might explain the link of CRC with obesity, and offers novel therapeutic and preventive perspectives in this specific clinical setting.

Peritoneal metastases (PM) are common routes of dissemination for colorectal cancer (CRC) and remain a lethal disease with a poor prognosis. The properties of the extracellular matrix (ECM) are important in cancer development; studying their changes is crucial to understand CRC-PM development. We studied the elastic properties of ECMs derived from human samples of normal and neoplastic PM by atomic force microscopy (AFM); results were correlated with patient clinical data and expression of ECM components related to metastatic spread. We show that PM progression is accompanied by stiffening of the ECM, increased cancer associated fibroblasts (CAF) activity and increased deposition and crosslinking in neoplastic matrices; on the other hand, softer regions are also found in neoplastic ECMs on the same scales. Our results support the hypothesis that local changes in the normal ECM can create the ground for growth and spread from the tumour of invading metastatic cells. We have found correlations between the mechanical properties (relative stiffening between normal and neoplastic ECM) of the ECM and patients’ clinical data, like age, sex, presence of protein activating mutations in BRAF and KRAS genes and tumour grade. Our findings suggest that the mechanical phenotyping of PM-ECM has the potential to predict tumour development.