Rapid autopsies to enhance metastatic research: the UPTIDER post-mortem tissue donation program.

Research on metastatic cancer has been hampered by limited sample availability. Here we present the breast cancer post-mortem tissue donation program UPTIDER and show how it enabled sampling of a median of 31 (range: 5-90) metastases and 5-8 liquids per patient from its first 20 patients. In a dedicated experiment, we show the mild impact of increasing time after death on RNA quality, transcriptional profiles and immunohistochemical staining in tumor tissue samples. We show that this impact can be counteracted by organ cooling. We successfully generated ex vivo models from tissue and liquid biopsies from distinct histological subtypes of breast cancer. We anticipate these and future findings of UPTIDER to elucidate mechanisms of disease progression and treatment resistance and to provide tools for the exploration of precision medicine strategies in the metastatic setting.

Retrospective Evaluation of the Prognostic Value of Histological Growth Pattern in Patients with Colorectal Peritoneal Metastases Undergoing Curative-Intent Cytoreductive Surgery.

BACKGROUND: Two distinct histological growth patterns (HGPs) were described in patients with peritoneal metastasis of colorectal cancer origin (PMCRC) with limited Peritoneal Cancer Index (PCI) ≤ 6 who did not receive neoadjuvant chemotherapy (NAC) and were treated with cytoreductive surgery (CRS) ± hyperthermic intraperitoneal chemotherapy (HIPEC): pushing HGP (P-HGP) and infiltrating HGP (I-HGP). Patients with dominant P-HGP (> 50%) had significantly better disease-free survival (DFS) and overall survival (OS). OBJECTIVE: We aimed to determine whether these previous observations regarding the prognostic value of HGP in patients with PMCRC with low PCI (≤ 6) are also valid in all operable patients, regardless of whether they received NAC or not and regardless of PCI score. METHODS: This was a retrospective study including 76 patients who underwent complete CRS ± HIPEC for PMCRC between July 2012 and March 2019. In each patient, up to five of the largest excised peritoneal nodules were analyzed for their tumor-to-peritoneum interface. Correlations between NAC, HGP, and prognosis were further explored. RESULTS: Thirty-seven patients (49%) had dominant P-HGP and 39 (51%) had dominant I-HGP. On univariate analysis, patients with P-HGP ≤ 50% had significantly lower OS than those with dominant P-HGP > 50% (39 versus 60 months; p = 0.014) confirmed on multivariate analysis (hazard ratio 2.4, 95% confidence interval 1.3-4.5; p = 0.006). There were no significant associations between NAC and type of HGP. CONCLUSIONS: This study confirms the prognostic value and reproducibility of the two previously reported HGPs in PMCRC. Dominant P-HGP is associated with better DFS and OS in patients undergoing curative-intent CRS ± HIPEC compared with I-HGP, independently of the extent of peritoneal disease burden.

Transcriptomic characterization of the histopathological growth patterns in breast cancer liver metastases.

Metastatic breast cancer (mBC) remains incurable and liver metastases (LM) are observed in approximately 50% of all patients with mBC. In some cases, surgical resection of breast cancer liver metastases (BCLM) is associated with prolonged survival. However, there are currently no validated marker to identify these patients. The interactions between the metastatic cancer cells and the liver microenvironment result in two main histopathological growth patterns (HGP): replacement (r-HGP), characterized by a direct contact between the cancer cells and the hepatocytes, and desmoplastic (d-HGP), in which a fibrous rim surrounds the tumor cells. In patients who underwent resection of BCLM, the r-HGP is associated with a worse postoperative prognosis than the d-HGP. Here, we aim at unraveling the biological differences between these HGP within ten patients presenting both HGP within the same metastasis. The transcriptomic analyses reveal overexpression of genes involved in cell cycle, DNA repair, vessel co-option and cell motility in r-HGP while angiogenesis, wound healing, and several immune processes were found overexpressed in d-HGP LM. Understanding the biology of the LM could open avenues to refine treatment of BC patients with LM.

Long-Term Results of a Phase 1 Dose Escalation Trial of Ablative Stereotactic Body Radiation Therapy.

PURPOSE: Stereotactic body radiation therapy is increasingly used for oligometastatic disease as well as palliation, but treatment protocols for nonspine bone and nodal metastases are lacking, with a wide variety of schedules applied. METHODS AND MATERIALS: A prospective dose-escalation trial was initiated, involving 90 patients, among whom 52 (58%) had primary prostate tumors, 13 had breast tumors (14%), and 25 (28%) had other primary tumor types. All visible lymph node or nonspine bone oligometastases were treated in 3 consecutive cohorts: 5 × 7.0 Gy, 3 × 10.0 Gy, or 1 × 20.0 Gy. RESULTS: Initial results revealed no dose-limiting toxicity after a median follow-up of 17.2 months. This update provides information on long-term toxicity, local failure (LF), and progression-free survival (PFS). After a median follow-up of 50 months, no new safety signals were observed. Grade 2 toxicity was 13%, 7% and 10% in the respective cohorts (P = .9), without grade 3 to 5 toxicities. LF rates were 9%, 3%, and 6% (P = .5) for the respective treatment groups, with an overall cumulative risk of LF of 7% (95% CI, 2-12) at 4 years. Median PFS was 16.5 months (95% CI, 9.8-21.5), and 4-year PFS was 21% (95% CI, 14-32). Median overall survival across groups was not reached (95% CI, 52.8 - not reached), 4-year OS was 68% (95% CI, 59-78). A subset of patients (23%) remained long-term disease-free, 37% had oligoprogressive disease at first recurrence and 40% developed polymetastatic relapse. CONCLUSIONS: The safe and effective use of dose-escalated single-fraction stereotactic body radiation therapy for bone and lymph node metastases is supported by this trial, especially considering patient-convenience and cost-effectiveness. Caution is needed when generalizing these outcomes beyond breast and prostate cancer, given their underrepresentation in our study.

Treatment Response, Tumor Infiltrating Lymphocytes and Clinical Outcomes in Inflammatory Breast Cancer-Treated with Neoadjuvant Systemic Therapy.

Inflammatory breast cancer (IBC) is a rare (1%-5%), aggressive form of breast cancer, accounting for approximately 10% of breast cancer mortality. In the localized setting, standard of care is neoadjuvant chemotherapy (NACT) ± anti-HER2 therapy, followed by surgery. Here we investigated associations between clinicopathologic variables, stromal tumor-infiltrating lymphocytes (sTIL), and pathologic complete response (pCR), and the prognostic value of pCR. We included 494 localized patients with IBC treated with NACT from October 1996 to October 2021 in eight European hospitals. Standard clinicopathologic variables were collected and central pathologic review was performed, including sTIL. Associations were assessed using Firth logistic regression models. Cox regressions were used to evaluate the role of pCR and residual cancer burden (RCB) on disease-free survival (DFS), distant recurrence-free survival (DRFS), and overall survival (OS). Distribution according to receptor status was as follows: 26.4% estrogen receptor negative (ER-)/HER2-; 22.0% ER-/HER2+; 37.4% ER+/HER2-, and 14.1% ER+/HER2+. Overall pCR rate was 26.3%, being highest in the HER2+ groups (45.9% for ER-/HER2+ and 42.9% for ER+/HER2+). sTILs were low (median: 5.3%), being highest in the ER-/HER2- group (median: 10%). High tumor grade, ER negativity, HER2 positivity, higher sTILs, and taxane-based NACT were significantly associated with pCR. pCR was associated with improved DFS, DRFS, and OS in multivariable analyses. RCB score in patients not achieving pCR was independently associated with survival. In conclusion, sTILs were low in IBC, but were predictive of pCR. Both pCR and RCB have an independent prognostic role in IBC treated with NACT. SIGNIFICANCE: IBC is a rare, but very aggressive type of breast cancer. The prognostic role of pCR after systemic therapy and the predictive value of sTILs for pCR are well established in the general breast cancer population; however, only limited information is available in IBC. We assembled the largest retrospective IBC series so far and demonstrated that sTIL is predictive of pCR. We emphasize that reaching pCR remains of utmost importance in IBC.

A flexible, image-based, high-throughput platform encompassing in-depth cell profiling to identify broad-spectrum coronavirus antivirals with limited off-target effects.

The recent pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) posed a major threat to global health. Although the World Health Organization ended the public health emergency status, antiviral drugs are needed to address new variants of SARS-CoV-2 and future pandemics. To identify novel broad-spectrum coronavirus drugs, we developed a high-content imaging platform compatible with high-throughput screening. The platform is broadly applicable as it can be adapted to include various cell types, viruses, antibodies, and dyes. We demonstrated that the antiviral activity of compounds against SARS-CoV-2 variants (Omicron BA.5 and Omicron XBB.1.5), SARS-CoV, and human coronavirus 229E could easily be assessed. The inclusion of cellular dyes and immunostaining in combination with in-depth image analysis enabled us to identify compounds that induced undesirable phenotypes in host cells, such as changes in cell morphology or in lysosomal activity. With the platform, we screened ∼900K compounds and triaged hits, thereby identifying potential candidate compounds carrying broad-spectrum activity with limited off-target effects. The flexibility and early-stage identification of compounds with limited host cell effects provided by this high-content imaging platform can facilitate coronavirus drug discovery. We anticipate that its rapid deployability and fast turnaround can also be applied to combat future pandemics.

Histopathological growth patterns and tumor-infiltrating lymphocytes in breast cancer liver metastases.

Liver is the third most common organ for breast cancer (BC) metastasis. Two main histopathological growth patterns (HGP) exist in liver metastases (LM): desmoplastic and replacement. Although a reduced immunotherapy efficacy is reported in patients with LM, tumor-infiltrating lymphocytes (TIL) have not yet been investigated in BCLM. Here, we evaluate the distribution of the HGP and TIL in BCLM, and their association with clinicopathological variables and survival. We collect samples from surgically resected BCLM (n = 133 patients, 568 H&E sections) and post-mortem derived BCLM (n = 23 patients, 97 H&E sections). HGP is assessed as the proportion of tumor liver interface and categorized as pure-replacement ('pure r-HGP') or any-desmoplastic ('any d-HGP'). We score the TIL according to LM-specific guidelines. Associations with progression-free (PFS) and overall survival (OS) are assessed using Cox regressions. We observe a higher prevalence of 'any d-HGP' (56%) in the surgical samples and a higher prevalence of 'pure r-HGP' (83%) in the post-mortem samples. In the surgical cohort, no evidence of the association between HGP and clinicopathological characteristics is observed except with the laterality of the primary tumor (p value = 0.049) and the systemic preoperative treatment before liver surgery (p value = .039). TIL is less prevalent in 'pure r-HGP' as compared to 'any d-HGP' (p value = 0.001). 'Pure r-HGP' predicts worse PFS (HR: 2.65; CI: (1.45-4.82); p value = 0.001) and OS (HR: 3.10; CI: (1.29-7.46); p value = 0.011) in the multivariable analyses. To conclude, we demonstrate that BCLM with a 'pure r-HGP' is associated with less TIL and with the worse outcome when compared with BCLM with 'any d-HGP'. These findings suggest that HGP could be considered to refine treatment approaches.

Histopathological growth pattern of liver metastases as an independent marker of metastatic behavior in different primary cancers.

Surgical resection can lead to prolonged survival in patients with isolated liver metastases (LM) from various primary cancers. However, there are currently no validated predictive markers to discriminate between these oligo/argometastatic patients, who will benefit from surgery, and those with diffuse metastatic behavior in whom surgery will be futile. To evaluate whether the tumor microenvironment, or histopathological growth pattern (HGP), of LM reflects the type of metastatic progression independently of the origin of the primary cancer, we analyzed a combined series of patients who underwent surgery for colorectal LM (N=263) or non-colorectal LM (N=66). HGPs of LM were scored in each patient to distinguish between desmoplastic HGP (all LM showing a complete encapsulated pattern) and non-desmoplastic HGP (at least one LM with some infiltrating-replacement component). In the entire series, 5-year overall and progression-free survival were, 44.5% and 15.5%, respectively, with no significant differences between colorectal and non-colorectal LM. In patients with desmoplastic HGP, 5-year overall and progression-free survival were 57% and 32%, respectively, as compared to 41% and 12%, respectively, in patients with non-desmoplastic-HGP (p=0.03 and 0.005). Irrespective of cancer origin and compared to traditional risk factors, desmoplastic HGP was the most significant predictor for better post-operative overall survival (adjusted HR: 0.62; 95% CI: [0.49-0.97]; p=0.035) and progression-free survival (adjusted HR: 0.61; 95% CI: [0.42-0.87], p=0.006). This suggests that the HGP of LM may represent an accurate marker that reflects the mode of metastatic behavior, independently of primary cancer type.

An idiosyncratic zonated stroma encapsulates desmoplastic liver metastases and originates from injured liver.

A perimetastatic capsule is a strong positive prognostic factor in liver metastases, but its origin remains unclear. Here, we systematically quantify the capsule's extent and cellular composition in 263 patients with colorectal cancer liver metastases to investigate its clinical significance and origin. We show that survival improves proportionally with increasing encapsulation and decreasing tumor-hepatocyte contact. Immunostaining reveals the gradual zonation of the capsule, transitioning from benign-like NGFRhigh stroma at the liver edge to FAPhigh stroma towards the tumor. Encapsulation correlates with decreased tumor viability and preoperative chemotherapy. In mice, chemotherapy and tumor cell ablation induce capsule formation. Our results suggest that encapsulation develops where tumor invasion into the liver plates stalls, representing a reparative process rather than tumor-induced desmoplasia. We propose a model of metastases growth, where the efficient tumor colonization of the liver parenchyma and a reparative liver injury reaction are opposing determinants of metastasis aggressiveness.

Association of body mass index with clinicopathological features and survival in patients with primary invasive lobular breast cancer.

PURPOSE: Invasive lobular carcinoma (ILC) represents up to 15% of all breast carcinomas. While the proportion of women with overweight and obesity increases globally, the impact of body mass index (BMI) at primary diagnosis on clinicopathological features of ILC and the prognosis of the patients has not been investigated yet. PATIENTS AND METHODS: We performed a multicentric retrospective study including patients diagnosed with non-metastatic pure ILC. The association of BMI at diagnosis with clinicopathological variables was assessed using linear or multinomial logistic regression. Univariable and multivariable survival analyses were performed to evaluate the association of BMI with disease-free survival (DFS), distant recurrence-free survival (DRFS), and overall survival (OS). RESULTS: The data of 2856 patients with ILC and available BMI at diagnosis were collected, of which 2570/2856 (90.0%) had oestrogen receptor (ER)-positive and human epidermal growth factor receptor (HER2) not amplified/overexpressed (ER+/HER2-) ILC. Of these 2570 patients, 80 were underweight (3.1%), 1410 were lean (54.9%), 712 were overweight (27.7%), and 368 were obese (14.3%). Older age at diagnosis, a higher tumour grade, a larger tumour size, a nodal involvement, and multifocality were associated with a higher BMI. In univariable models, higher BMI was associated with worse outcomes for all end-points (DFS: hazard ratio (HR) 1.21, 95CI 1.12-1.31, p value<0.01; DRFS: HR 1.25, 95CI 1.12-1.40, p value<0.01; OS: HR 1.25, 95CI 1.13-1.37, p value<0.01). This association was not statistically significant in multivariable analyses (DFS: HR 1.09, 95CI 0.99-1.20, p value 0.08; DRFS: HR 1.03, 95CI 0.89-1.20, p value 0.67; OS: HR 1.11, 95CI 0.99-1.24, p value 0.08), whereas grade, tumour size, and nodal involvement were still prognostic for all end-points. CONCLUSION: Worse prognostic factors such as higher grade, larger tumour size, and nodal involvement are associated with higher BMI in ER+/HER2- ILC, while there was no statistical evidence for an independent prognostic role for BMI. Therefore, we hypothesise that the effect of BMI on survival could be mediated through its association with these clinicopathological variables.

Immunohistochemical characterisation of the immune landscape in primary uveal melanoma and liver metastases.

BACKGROUND: The immune landscape of uveal melanoma liver metastases (UMLM) has not been sufficiently studied. METHODS: Immune cell infiltrates (ICIs), PD-1 and PD-L1 were characterised in 62 UMLM and 28 primary uveal melanomas (PUM). ICI, PD-1 and PD-L1 were scored as: (1) % tumoral area occupied by tumour-infiltrating lymphocytes or macrophages (TILs, TIMs) and (2) % perTumoral (perT) area. ICIs and other variables including histopathologic growth patterns (HGPs), replacement and desmoplastic, of UMLM were analysed for their prognostic value. RESULTS: ICIs recognised by haematoxylin-eosin-saffron (HES) and IHC (e.g., T cells (CD3), B cells (CD20). Macrophages (CD68), (CD163), were primarily localised to the perT region in PUM and UMLM and were more conspicuous in UMLM. HES, CD3, CD4, FoxP3, CD8, CD20, PD-1 TILs were scant (<5%). TIMs were more frequent, particularly in UMLM than in PUM. Both CD68+ TIMs and HGPs remained significant on multivariate analysis, influencing overall (OS) and metastasis-specific overall survival (MSOS). CD68 + , CD163+ and CD20+ perT infiltrates in UMLM predicted increased OS and MSOS on univariate analysis. CONCLUSIONS: TILs and PD-L1 have no predictive value in PUM or UMLM. CD68+ and CD163+TIMs, CD20+ perT lymphocytes, and HGPs are important prognostic factors in UMLMs.

Association between primary tumor characteristics and histopathological growth pattern of liver metastases in colorectal cancer.

INTRODUCTION: The microarchitecture of liver metastases (LMs), or histopathological growth pattern (HGP), has been demonstrated to be a significant prognostic factor in patients undergoing resection of colorectal liver metastases (CRLMs). Currently, however, HGP can be only determined on the operative specimen. Therefore, the development of new tools to predict the HGP of CRLMs before surgery and to understand the mechanisms that drive these patterns is important for improving individualization of therapeutic management. In this study, we analyzed data from a retrospective series of patients who underwent surgery for CRLMs to compare primary tumor characteristics, including markers of local aggressiveness and migratory capacity, and HGP of liver metastases. METHODS: Data from a retrospective series of 167 patients who underwent curative-intent resection of CRLMs and in whom pathological samples from both primary tumor and liver metastases were available were reviewed. At the primary tumor level, KRAS mutational status, grade of differentiation, and tumor budding were assessed. HGP was scored in each resected CRLM, according to consensus guidelines, and classified as desmoplastic (dHGP) or non-desmoplastic (non-dHGP). Associations between primary tumor characteristics and HGP of CRLMs were evaluated using a binary logistic regression model. Overall survival and disease-free survival were evaluated using Kaplan-Meier and multivariable Cox regression analyses. RESULTS: CRLMs were classified as dHGP in 36% of the patients and as non-dHGP in 64%. Higher rates of moderately or poorly differentiated primary tumors were observed in the non-dHGP CRLM group (80%), as compared with the dHGP group (60%) (OR = 3.6; 95%CI: 1.6-7.05; p = 0.001). Higher rates of tumor budding were observed in the non-dHGP CRLM group, with a median tumor budding value of 4 as compared with 2.5 in the dHGP group (p = 0.042). In the entire series, 5-year overall and disease-free survival were 43% and 32.5%, respectively. The non-dHGP CRLM group had worse post-hepatectomy survival, with 5-year overall and disease-free survival of 32.2% and 24.6%, respectively, as compared with 60.8% and 45.9%, respectively, for the dHGP group (p = 0.02). CONCLUSION: Colorectal tumors with moderate or poor differentiation and those with high tumor budding are more frequently associated with CRLMs with a non-dHGP. This suggests that primary tumor characteristics of local aggressiveness and migratory capacity could preferentially promote the development of CRLMs with an infiltrating pattern and that these parameters should be considered as part of new scores for predicting HGP before surgery. This finding may stimulate new lines of research for more individualized therapeutic decision in patients with CRLM candidate to surgery.

The Prognostic Value of Distinct Histological Growth Patterns of Colorectal Peritoneal Metastases: A Pilot Study.

BACKGROUND: Different histological growth patterns (HGP) describing the tumor-to-liver interface have been described in colorectal liver metastases and have been associated with a strong prognostic value. However, HGP of peritoneal metastases (PM) of colorectal cancer (CRC) have not yet been described. Our objective was to determine whether distinct HGP can be identified in PMCRC and to evaluate their potential prognostic value in these patients. METHODS: This retrospective study included 38 patients who underwent curative-intent surgery for PMCRC between July 2012 and March 2019, with PCI≤6, and who had not received preoperative chemotherapy. In each patient, the tumor-to-peritoneum interface was evaluated in the excised peritoneal nodules. The association between HGP and postoperative survival was analyzed by using the Kaplan-Meier method. RESULTS: Two distinct HGP were identified: a pushing-type (P-HGP), characterized by a fibrous rim separating the PM and peritoneum, and an infiltrating-type (I-HGP), characterized by focal penetration of tumor cells into the surrounding peritoneal lining without a fibrous rim. Fifteen patients had dominant P-HGP, and 23 patients had dominant I-HGP. Patients with dominant P-HGP (>50% tumor-peritoneum interface) had a significantly better DFS (30 months) than those with P-HGP <50% (9 months; p = 0.029). Patients with a P-HGP dominance >60% had better OS (131 months) than those with P-HGP <60% (41 months; p = 0.044). CONCLUSIONS: This is the first description of two distinct, reproducible HGP in PMCRC. The dominant P-HGP is associated with a favorable prognosis in patients with PMCRC, compared with I-HGP, suggesting that this parameter could ultimately represent a new prognostic biomarker.

Molecular differences of angiogenic versus vessel co-opting colorectal cancer liver metastases at single-cell resolution.

BACKGROUND: Colorectal cancer liver metastases (CRCLM) are associated with a poor prognosis, reflected by a five-year survival rate of 14%. Anti-angiogenic therapy through anti-VEGF antibody administration is one of the limited therapies available. However, only a subgroup of metastases uses sprouting angiogenesis to secure their nutrients and oxygen supply, while others rely on vessel co-option (VCO). The distinct mode of vascularization is reflected by specific histopathological growth patterns (HGPs), which have proven prognostic and predictive significance. Nevertheless, their molecular mechanisms are poorly understood. METHODS: We evaluated CRCLM from 225 patients regarding their HGP and clinical data. Moreover, we performed spatial (21,804 spots) and single-cell (22,419 cells) RNA sequencing analyses to explore molecular differences in detail, further validated in vitro through immunohistochemical analysis and patient-derived organoid cultures. RESULTS: We detected specific metabolic alterations and a signature of WNT signalling activation in metastatic cancer cells related to the VCO phenotype. Importantly, in the corresponding healthy liver of CRCLM displaying sprouting angiogenesis, we identified a predominantly expressed capillary subtype of endothelial cells, which could be further explored as a possible predictor for HGP relying on sprouting angiogenesis. CONCLUSION: These findings may prove to be novel therapeutic targets to the treatment of CRCLM, in special the ones relying on VCO.

Comparison of the genomic alterations present in tumor samples from patients with metastatic inflammatory versus non-inflammatory breast cancer reveals AURKA as a potential treatment target.

Inflammatory breast cancer (IBC) is a rare but aggressive subtype of breast cancer, mainly characterized using primary tumor samples. Here, using public datasets, we compared the genomic alterations in primary and metastatic samples from patients with metastatic IBC versus patients with metastatic non-IBC. We observed a higher frequency of AURKA amplification in IBC. We further showed that AURKA amplification was associated with increased AURKA mRNA expression, which we demonstrated was higher in IBC. Finally, higher protein expression of AURKA was associated with worse prognosis in patients with IBC. These findings deserve further investigation given the existence of AURKA-inhibitors.

Tumor biology reflected by histological growth pattern is more important than surgical margin for the prognosis of patients undergoing resection of colorectal liver metastases.

INTRODUCTION: The histological growth pattern (HGP) of colorectal liver metastases (CRLMs) reflects tumor biology and local infiltrating behavior. In patients undergoing surgery for CRLMs, we investigated whether HGP and surgical margin status interact when influencing prognosis. METHODS: Clinicopathological data, margin status, and HGP were reviewed in patients who underwent resection of CRLMs. R1 margin was defined when cancer cells were present at any point along the margin. HGPs were scored according to international guidelines, identifying patients with desmoplastic (DHGP) or non-desmoplastic (non-DHGP) CRLMs. RESULTS: Among 299 patients, 16% had R1 resection and 81% had non-DHGP CRLMs. Non-DHGP was the only predictive factor for R1 resection (18.7% versus 7.4% in DHGP, p = 0.04). Poorer 5-year overall survival was observed in both R1 and non-DHGP groups in univariate analysis (27.6% in R1 versus 45.6% in R0, p = 0.026, and 37.2% in non-DHGP versus 59.2% in DHGP, p = 0.013), whereas non-DHGP but not R1 remained associated with worse prognosis in multivariate analysis. In patients with non-DHGP, R1 margin has no prognostic impact. CONCLUSIONS: In patients undergoing resection of CRLMs, the prognostic value of poor tumor biology, such as in patients with non-DHGP, exceeds that of surgical radicality.

Generation of vessel co-option lung metastases mouse models for single-cell isolation of metastases-derived cells and endothelial cells.

Tumor vessel co-option, a process in which cancer cells "hijack" pre-existing blood vessels to grow and invade healthy tissue, is poorly understood but is a proposed resistance mechanism against anti-angiogenic therapy (AAT). Here, we describe protocols for establishing murine renal (RENCA) and breast (4T1) cancer lung vessel co-option metastases models. Moreover, we outline a reproducible protocol for single-cell isolation from murine lung metastases using magnetic-activated cell sorting as well as immunohistochemical stainings to distinguish vessel co-option from angiogenesis. For complete details on the use and execution of this protocol, please refer to Teuwen et al. (2021).