Therapy Testing in a Spheroid-based 3D Cell Culture Model for Head and Neck Squamous Cell Carcinoma

Summary

We describe the evolution of a spheroid-based, three-dimensional in vitro model that enables us to test the current standard of experimental therapy regimens for head and neck squamous cell carcinoma on cell lines, aiming at evaluating therapy susceptibility and resistance on primary cells from human specimens in the future.

Abstract

Current treatment options for advanced and recurrent head and neck squamous cell carcinoma (HNSCC) enclose radiation and chemo-radiation approaches with or without surgery. While platinum-based chemotherapy regimens currently represent the gold standard in terms of efficacy and are given in the vast majority of cases, new chemotherapy regimens, namely immunotherapy are emerging. However, the response rates and therapy resistance mechanisms for either chemo regimen are hard to predict and remain insufficiently understood. Broad variations of chemo and radiation resistance mechanisms are known to date. This study describes the development of a standardized, high-throughput in vitro assay to assess HNSCC cell line's response to various therapy regimens, and hopefully on primary cells from individual patients as a future tool for personalized tumor therapy. The assay is designed to being integrated into the quality-controlled standard algorithm for HNSCC patients at our tertiary care center; however, this will be subject of future studies. Technical feasibility looks promising for primary cells from tumor biopsies from actual patients. Specimens are then transferred into the laboratory. Biopsies are mechanically separated followed by enzymatic digestion. Cells are then cultured in ultra-low adhesion cell culture vials that promote the reproducible, standardized and spontaneous formation of three-dimensional, spheroid-shaped cell conglomerates. Spheroids are then ready to be exposed to chemo-radiation protocols and immunotherapy protocols as needed. The final cell viability and spheroid size are indicators of therapy susceptibility and therefore could be drawn into consideration in future to assess the patients' likely therapy response. This model could be a valuable, cost-efficient tool towards personalized therapy for head and neck cancer.

Introduction

Head and Neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide with a rising incidence of mucosal human papillomavirus (HPV) infection-associated pathogenesis, next to a majority of cases caused by excessive nicotine and alcohol consumption ^1,2. While smaller tumors and pre-invasive stages are usually well treatable with surgical excision, usually combined with cervical lymph node dissection, treatment for advanced-stage and recurrent HNSCC remains challenging due to aggressive tumor invasion with metastatic spread and resistance to radiation and chemotherapy protocols

Protocol

All studies shown in this manuscript, namely the use of human tumor specimens, are protected under and in consent with prior decisions from University Medicine of Mainz/University of Munich Medical Center Ethics committee. Patients have given informed consent according to national legal guidelines agreeing to scientific use of excess biological material that was obtained in the course of their treatment. Research has been performed in compliance with all institutional, national and international guidelines for human welfare.

1. Taking a Tumor Biopsy from Head and Heck Squamous Cell Carcinoma

1. Perform general (propofol and/or sevo....

Results

We were able to reproducibly generate spheroids from single cell suspensions, first from different cell lines including the proprietary PiCa cell line, later from primary human cancer cells derived from fresh tumor biopsies as described in Hagemann et al.²⁶. We evaluated two established methods for spheroid generation; the two being the so-called hanging drop (HD) method and the ultra-low adhesion (ULA) method, the latter being the more effective and safe .......

Discussion

We were able to establish a protocol to generate reproducible spheroids from cell suspensions, for both cell lines and, in preliminary experiments, primary human tumor cells. We first assessed two previously described methods and identified the ULA-method, a method where culture plates with ultra-low adhesion surfaces are used, to be the safer and more reliable one for the generation of uniform three-dimensional spheroids. By combining two separate methods for assay read-out (size/area and cell viability), this multimoda.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Dulbeccos modified Eagles medium (DMEM)	Biochrom, Berlin, Germany	F 0425
Fetal bovine serum	Gibco Life Technologies, Paisley, UK	10500-064
penicillin/streptomycin	Biochrom, Berlin, Germany	A2212
sodium pyruvate	Biochrom, Berlin, Germany	L0473
non-essential amino acids	Biochrom, Berlin, Germany	K0293
L-Glutamine	Biochrom, Berlin, Germany	K0293
Liberase	Roche Life Sciences, Basel, Switzerland	5401127001
GravityPLUS 3D Culture and Assay Platform	InSphero, Schlieren, Switzerland	PB-CS 06-001
GravityTRAP plate	InSphero, Schlieren, Switzerland	PB-CS-01-001
Ultra-low attachment (ULA) culture plates	Corning, Corning, NY, USA	4520
airway epithelial cell growth medium	Promocell, Heidelberg, Germany	C-21060
amphotericin B	Biochrom, Berlin, Germany	A 2612
airway epithelial cell growth medium supplement mix	Promocell, Heidelberg, Germany	C39165
WST-8 test	Promocell, Heidelberg, Germany	PC PK-CA705-CK04
Keratinocyte SFMedium + L-Glutamine 500mL	Invitrogen	#17005-034
Bovine Pituitary Extract (BPE), 25mg	Invitrogen	#37000015
Recombinant human Epithelial Growth Factor 2.5 µg	Invitrogen	#37000015
DMEM High Glucose	Invitrogen	#21068-028
Penicillin Streptomycin 10000U/mL Penicillin/ 10000µg/mL Streptomycin	Invitrogen	#15140-122
F12 Nutrient Mix	Invitrogen	#21765-029
Glutamax (200 mM L-Alanyl-L-Glutamin-Dipeptide in NaCl)	Invitrogen	#35050087
HBSS (Ca, Mg)	Life Technologies	#14025-092	(no phenol red)
1x TrypLE Expres Enzyme	Invitrogen	#12604-013	(no phenol red)
Accutase (enzymatic cell detachment solution)	Innovative cell technologies	Cat# AT104
70 µm Falcon cell strainer	BD Biosciences, USA	#352350