Colorectal cancer (CRC) is the third most commonly diagnosed malignancy, as well as the second leading cause of cancer-related mortality worldwide. Most of CRC patients develop colorectal liver metastases (CLM), for whom hepatic resection combined with systemic chemotherapy may be associated with increased overall survival rates. Nevertheless, in the era of precision medicine there is a critical need of prognostic markers to cope with the heterogeneity of CLM patients. In the last years, the field of cancer immunology has caught the attention of surgical and medical oncologist and immune-based anticancer strategies have been applied to different clinical scenarios with promising results. Among inflammatory cells, tumor-associated macrophages (TAMs) pave the way to tissue invasion and intravasation providing a nurturing microenvironment for metastases. The quantification of immune landscape of tumors has provided novel prognostic indicators of cancer progression, and the quantification of TAMs might explain the heterogeneity of CLM patients. Previous studies on this field demonstrated that morphology of tumour-associated macrophages (TAMs) correlates with prognosis in CLM patients. Specifically, while density of TAMs does not correlate with survival of CLM patients, the area of TAMs dictated the prognosis of these patients, with smaller TAMs conferring a better prognosis than larger ones. Starting from these results, the aim of this thesis project is to characterize the diversity of TAMs features in CLM and to investigate their role in tumor characteristics, to different chemotherapy regimens and whether they can be used as biomarkers to promote an individualized therapeutic approach for CLM patients. The research project has been carried out at Hepatobiliary Immunopathology Laboratory, Humanitas Clinical and Research Center (Rozzano, Italy), led by Professor Ana Lleo. Samples from CLM peritumoral, tumoral liver tissue and peripheral blood were collected from of CLM patients, both when operated simultaneously or when operated separately on primary and metastatic sites of disease. Patients were operated by the proposed PI at the Department of Hepatobiliary and General Surgery, Humanitas University, IRCCS Humanitas Research Hospital. From each single patient, immune infiltrate cell suspensions for subsequent analysis were obtained through collagenase IV enzymatic digestion of tissue samples. On the basis of the evidence that TAMs morphology correlate with distinct clinical outcomes, deep phenotyping studies of TAMs in CLM were performed by multi-parametric flow cytometry. Besides, to appreciate TAMs heterogeneity in terms of size and morphology and correlate them with flow cytometry data, FFPE specimens collected from peritumoral tissues of CLM patients were analyzed by immunohistochemistry. On a small subset of CLM patients, TAMs were sorted separately according to morphological features and subsequently characterized by gene expression profiling through qRT-PCR, aiming to provide more precise functional characterization of TAMs correlated with their size. Finally, all the data collected were correlated with tumor characteristics, aggressiveness, patient treatment and survival. The expected findings may allow to develop a new diagnostic tool based on TAMs features, which can open the way to new criteria for patient stratification and for the design of new targeted therapies in a personalized medicine perspective.
Il cancro colon-rettale è il terzo tumore più comunemente diagnosticato e la seconda causa di morte per cancro al mondo. La maggior parte dei pazienti affetti da questo tipo di tumore vanno incontro a metastasi localizzate nel fegato, il cui trattamento comprende resezione epatica combinata con chemioterapia, che possono portare ad un incremento delle possibilità di sopravvivenza. Tuttavia, nell’era della medicina di precisione, è impellente la necessità di marker prognostici in grado di far fronte all’eterogeneità dei pazienti affetti da questa tipologia di metastasi epatiche. Negli ultimi anni, il campo dell’immunologia nei tumori ha catturato l’attenzione di medici e oncologi e le strategie anticancro basate sull’immunoterapia sono state applicate in vari scenari clinici mostrando risultati promettenti. Tra le varie tipologie di cellule infiammatorie, i macrofagi tumore-associati (TAMs) aprono la strada alle metastasi, promuovendo l’invasione dei tessuti e dei vasi sanguigni e linfatici. La quantificazione dell’infiltrato immunitario ha portato alla scoperta di nuovi indicatori prognostici della progressione tumorale, e la quantificazione dei macrofagi associati al tumore potrebbe aiutare a chiarire l’eterogeneità dei pazienti affetti da metastasi epatiche da cancro al colon-retto. Studi precedenti effettuati in questo campo hanno mostrato inoltre che la morfologia dei macrofagi associati al tumore è correlata alla prognosi dei pazienti, con una minore progressione della malattia in presenza di macrofagi di dimensioni ridotte, mentre in presenza di macrofagi di grandi dimensioni la prognosi risulta peggiore. Partendo da questi risultati, lo scopo di questa tesi è di caratterizzare la variazione delle caratteristiche dei macrofagi nelle metastasi epatiche da colon-retto e investigare il loro ruolo nel tumore, nella risposta ai differenti regimi chemioterapici e se possono essere utilizzati come marker per promuovere un approccio terapeutico individuale per i pazienti. Il progetto di ricerca è stato svolto nel Laboratorio di Immunopatologia Epatobiliare, Humanitas Clinical and Research Center (Rozzano, Italia), guidato dalla professoressa Ana Lleo. I campioni di fegato normale (localizzato lontano dal tumore), peritumorale e tumorale sono stati raccolti dai pazienti, operati simultaneamente o separatamente nel tumore primario e alle metastasi epatiche, presso il Dipartimento di Chirurgia Generale e Epatobiliare, Humanitas University, IRCCS Humanitas Research Hospital. Partendo dai campioni di ogni singolo paziente, sono state ottenute delle sospensioni di cellule di infiltrato immunitario di tessuto peritumorale tramite digestione enzimatica con l’enzima collagenasi di tipo IV. Basandoci sull’evidenza che la morfologia dei macrofagi associati al tumore è correlata alla prognosi dei pazienti, sono state svolte analisi fenotipiche approfondite dei macrofagi utilizzando citometria a flusso multi-parametrica. Inoltre, per apprezzare meglio la morfologia dei macrofagi e correlarla alle analisi svolte tramite citometria, sono state analizzate le sezioni di tessuto peritumorale dei pazienti tramite immunoistochimica. In un sottogruppo dei pazienti in analisi, i macrofagi sono stati sortati tramite cell sorting in accordo con le loro caratteristiche morfologiche e successivamente caratterizzati tramite qRT-PCR, con il fine di ottenere una caratterizzazione funzionale precisa dei macrofagi tumore-associati, correlandola con le caratteristiche del tumore, la sua aggressività, la risposta ai trattamenti e la sopravvivenza dei pazienti. I risultati ottenuti possono permettere di sviluppare un nuovo strumento diagnostico basato sulle caratteristiche dei macrofagi tumore-associati, che possono spianare la strada per ottenere nuovi criteri per la stratificazione dei pazienti e per il design di nuove terapie mirate nella prospettiva di una futura medicina personalizzata per i pazienti.
Dissecting the role of tumour-associated macrophages as a new diagnostic tool to refine the prognosis of patients undergoing hepatic resection for colorectal liver metastases: a prospective and retrospective study.
GAZZILLO, AURORA
2021/2022
Abstract
Colorectal cancer (CRC) is the third most commonly diagnosed malignancy, as well as the second leading cause of cancer-related mortality worldwide. Most of CRC patients develop colorectal liver metastases (CLM), for whom hepatic resection combined with systemic chemotherapy may be associated with increased overall survival rates. Nevertheless, in the era of precision medicine there is a critical need of prognostic markers to cope with the heterogeneity of CLM patients. In the last years, the field of cancer immunology has caught the attention of surgical and medical oncologist and immune-based anticancer strategies have been applied to different clinical scenarios with promising results. Among inflammatory cells, tumor-associated macrophages (TAMs) pave the way to tissue invasion and intravasation providing a nurturing microenvironment for metastases. The quantification of immune landscape of tumors has provided novel prognostic indicators of cancer progression, and the quantification of TAMs might explain the heterogeneity of CLM patients. Previous studies on this field demonstrated that morphology of tumour-associated macrophages (TAMs) correlates with prognosis in CLM patients. Specifically, while density of TAMs does not correlate with survival of CLM patients, the area of TAMs dictated the prognosis of these patients, with smaller TAMs conferring a better prognosis than larger ones. Starting from these results, the aim of this thesis project is to characterize the diversity of TAMs features in CLM and to investigate their role in tumor characteristics, to different chemotherapy regimens and whether they can be used as biomarkers to promote an individualized therapeutic approach for CLM patients. The research project has been carried out at Hepatobiliary Immunopathology Laboratory, Humanitas Clinical and Research Center (Rozzano, Italy), led by Professor Ana Lleo. Samples from CLM peritumoral, tumoral liver tissue and peripheral blood were collected from of CLM patients, both when operated simultaneously or when operated separately on primary and metastatic sites of disease. Patients were operated by the proposed PI at the Department of Hepatobiliary and General Surgery, Humanitas University, IRCCS Humanitas Research Hospital. From each single patient, immune infiltrate cell suspensions for subsequent analysis were obtained through collagenase IV enzymatic digestion of tissue samples. On the basis of the evidence that TAMs morphology correlate with distinct clinical outcomes, deep phenotyping studies of TAMs in CLM were performed by multi-parametric flow cytometry. Besides, to appreciate TAMs heterogeneity in terms of size and morphology and correlate them with flow cytometry data, FFPE specimens collected from peritumoral tissues of CLM patients were analyzed by immunohistochemistry. On a small subset of CLM patients, TAMs were sorted separately according to morphological features and subsequently characterized by gene expression profiling through qRT-PCR, aiming to provide more precise functional characterization of TAMs correlated with their size. Finally, all the data collected were correlated with tumor characteristics, aggressiveness, patient treatment and survival. The expected findings may allow to develop a new diagnostic tool based on TAMs features, which can open the way to new criteria for patient stratification and for the design of new targeted therapies in a personalized medicine perspective.È consentito all'utente scaricare e condividere i documenti disponibili a testo pieno in UNITESI UNIPV nel rispetto della licenza Creative Commons del tipo CC BY NC ND.
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https://hdl.handle.net/20.500.14239/14577