FBS-supplement based 2D cell culture has been considered until now the gold-standard methodology for growth of several cell culture lines. Nevertheless, the manufacturing of FBS has been associated with some issues, including exploitation of animals, batch-to-batch heterogenicity, and immunogenicity side effects. For this reason, several FBS-free alternatives have been developed overtime, including serum-based supplements (e.g., human serum and platelet-rich plasma). Among these, human platelet lysate (hPL) has been used successfully not only for the proliferation of several eukaryotic cell lines but also for increasing their growth, due to higher content of growth factors released from platelets, such as transforming growth factor-b (TGF-b) and vascular endothelial growth factor (VEGF). Here we optimized the gradual adaptation process of various cancer cell lines (A431, MDA-MB-231 and HCC-1937) in one of the commercialized hPL named Elarem™ Prime, produced by PL Bioscience, by gradually increasing the amount of platelet-lysate supplemented medium and decreasing the conventional FBS-supplemented medium (in which the immortalized cell lines had been grown). Different concentrations of Elarem Prime supplement were tested to assess which one could be used as substitute to FBS-based medium for cell proliferation. For this reason, doubling time and confluency studies were performed. Further studies may include the development of humanized 3D culture models (e.g., cancer-immune cell heterospheroids), where cell growth will be based on platelet-lysate-supplemented medium. Such practice may allow for a more suitable investigation of immune-cancer cell interactions, without any FBS-derived immunogenic side effects. After completing this first project, the second part of the experimental work was dedicated on identifying the immune cell pattern on triple negative breast cancer (TNBC)-derived lung metastasis. Since primary TNBC and metastasis are lacking apparent molecular targets, and therefore not responding to receptor-targeting therapeutic approaches (e.g., hormones), ex vivo analyses were conducted in order to identify alternative therapeutic targets. For achieving this, lungs with and without metastasis from Balb/c mice injected with 4T1 breast cancer cells had been isolated. These samples were subjected to histological analyses and optimization of immunofluorescence staining was performed to characterize the immune microenvironment of lung metastasis, derived from TNBC. Efforts have been particularly directed to the characterization of myeloid cell populations (including macrophages and neutrophils) as promising targets in the tumor microenvironment (TME). Our findings showed the presence of a particular cluster of over-expressed SiglecF+ myeloid cells inside the lung metastatic niche, together with the concomitant presence of F4/80+ macrophages. Further characterization, and expansion of the investigation to the lymphoid cell populations, may facilitate the development of new immune-cell targeting therapeutic strategies.
La coltura cellulare in 2D basata sull'utilizzo di siero fetale bovino (FBS) è fino ad ora ritenuta la metodologia per eccellenza per supportare la crescita di diverse linee cellulari. Nonostante ciò, sono stati associati diversi svantaggi conseguenti all'utilizzo di FBS come supplemento, inclusi uno sfruttamento degli animali, una eterogeneità batch-to-batch ed effetti indesiderati di carattere immunologico. Per questo motivo, alcune alternative cosiddette <<FBS-free>> sono state sviluppate nel tempo, inclusi supplementi basati sul siero (cfr. siero umano e plasma ricco in piastrine). Tra queste, il lisato di piastrine umano (hPL) è stato utilizzato con successo non solo per permettere la proliferazione di diverse linee cellulari procariotiche ma anche per incrementarne la crescita. Ciò è dovuto al più alto contenuto di fattori di crescita che vengono rilasciati dalle piastrine, come ad esempio il fattore trasformante di crescita (TGF-beta) ed il fattore di crescita dell'endotelio vascolare (VEGF). Nel caso in esame abbiamo ottimizzato il graduale processo di adattamento di diverse linee cellulari, una tumorale epidermoide (A431) e due epiteliali di carcinoma mammario triplo negativo (MDA-231 e HCC-1937) in un terreno di coltura addizionato con lisato di piastrine umano (cfr. nome commerciale Elarem™ Prime), prodotto dalla compagnia PL Bioscience di Aquisgrana. Questo processo è stato ottenuto attraverso diversi step che hanno visto l'utilizzo graduale di terreno di coltura addizionato con hPL mescolato con terreno di coltura convenzionale (senza però FBS). Concentrazioni differenti di Elarem Prime sono state testate per confrontare quale concentrazione potesse essere la migliore nel sostituire il terreno di coltura tradizionale basato sull'utilizzo di FBS. Per poter fare questa considerazione sono stati condotti studi di tempo di duplicazione ed osservazioni qualitative di confluenza cellulare. Studi successivi potrebbero includere il passaggio di queste cellule adattate ad un lisato di piastrine per un modello 3D (cfr. eterosferoidi) per poter effettuare studi di carattere immunologico ed assestare relazioni tra cellule cancerogene ed immunologiche di un modello tumorale (come il carcinoma mammario triplo negativo). Successivamente, nella seconda parte di questo lavoro sperimentale si è voluti dedicare nel caratterizzare il pattern immunologico nell'ambiente tumorale (TME) di metastasi polmonari derivanti dal tumore mammario triplo negativo. Quest'ultimo difatti è caratterizzato tuttora da una mancanza di targets molecolari e, quindi, spesso refrattario ad approcci terapeutici convenzionali, come ad esempio chemioterapia o trattamenti ormonali. La caratterizzazione immunologica è stata effettuata tramite analisi istologiche ex-vivo di metastasi polmonari murini derivanti dal tumore mammario triplo negativo (TNBC), indotto tramite inoculazione di cellule tumorali mammarie 4T1. Particolare attenzione è stata rivolta al compartimento mieloide (cfr. concetto di myeloid tuning) all'interno del microambiente tumorale. I nostri risultati hanno evidenziato la presenza di un particolare tipo di cluster pro-tumorale di cellule mieloidi (neutrofili positivi al marker SiglecF) presenti nell'ambiente circostante le infiltrazioni metastatiche, insieme alla presenza di macrofagi (identificati grazie al marker F4/80). La presenza di questa popolazione di neutrofili anche in infiltrazioni secondarie (e quindi non solo in tumori primari) può aprire il via allo sviluppo di una strategia terapeutica basata sull'utilizzare come target popolazioni immunitarie. Tra le considerazioni future potrebbe essere interessante anche studiare la relazione che intercorre tra popolazione mieloide e compartimento linfoide all'interno del microambiente tumorale.
Adaptation of cancer cell lines in platelet lysate-based medium and investigation of myeloid cell infiltration in murine TNBC-derived lung metastasis
FILOSA, ANNALISA
2021/2022
Abstract
FBS-supplement based 2D cell culture has been considered until now the gold-standard methodology for growth of several cell culture lines. Nevertheless, the manufacturing of FBS has been associated with some issues, including exploitation of animals, batch-to-batch heterogenicity, and immunogenicity side effects. For this reason, several FBS-free alternatives have been developed overtime, including serum-based supplements (e.g., human serum and platelet-rich plasma). Among these, human platelet lysate (hPL) has been used successfully not only for the proliferation of several eukaryotic cell lines but also for increasing their growth, due to higher content of growth factors released from platelets, such as transforming growth factor-b (TGF-b) and vascular endothelial growth factor (VEGF). Here we optimized the gradual adaptation process of various cancer cell lines (A431, MDA-MB-231 and HCC-1937) in one of the commercialized hPL named Elarem™ Prime, produced by PL Bioscience, by gradually increasing the amount of platelet-lysate supplemented medium and decreasing the conventional FBS-supplemented medium (in which the immortalized cell lines had been grown). Different concentrations of Elarem Prime supplement were tested to assess which one could be used as substitute to FBS-based medium for cell proliferation. For this reason, doubling time and confluency studies were performed. Further studies may include the development of humanized 3D culture models (e.g., cancer-immune cell heterospheroids), where cell growth will be based on platelet-lysate-supplemented medium. Such practice may allow for a more suitable investigation of immune-cancer cell interactions, without any FBS-derived immunogenic side effects. After completing this first project, the second part of the experimental work was dedicated on identifying the immune cell pattern on triple negative breast cancer (TNBC)-derived lung metastasis. Since primary TNBC and metastasis are lacking apparent molecular targets, and therefore not responding to receptor-targeting therapeutic approaches (e.g., hormones), ex vivo analyses were conducted in order to identify alternative therapeutic targets. For achieving this, lungs with and without metastasis from Balb/c mice injected with 4T1 breast cancer cells had been isolated. These samples were subjected to histological analyses and optimization of immunofluorescence staining was performed to characterize the immune microenvironment of lung metastasis, derived from TNBC. Efforts have been particularly directed to the characterization of myeloid cell populations (including macrophages and neutrophils) as promising targets in the tumor microenvironment (TME). Our findings showed the presence of a particular cluster of over-expressed SiglecF+ myeloid cells inside the lung metastatic niche, together with the concomitant presence of F4/80+ macrophages. Further characterization, and expansion of the investigation to the lymphoid cell populations, may facilitate the development of new immune-cell targeting therapeutic strategies.| File | Dimensione | Formato | |
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https://hdl.handle.net/20.500.12608/10167