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

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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^3,4,5,6,7,8. Recent studies suggest a high variability of cellular phenotype, and sub-characterization of circulating and disseminated tumor cells has just begun^9,10. The earlier belief of a solid, uniform tumor mass had to be revised in the light of recent studies in the past years^11,12,13,14. Current approaches for tumor characterization and identification of key mutations could identify several genes that seem to be associated with therapy resistance but remain a cost-intensive approach. Moreover, knowledge of genotype does not necessarily allow a reliable prediction of phenotype and its treatment response.

There have been few advances in improving overall and disease-free survival for advanced-stage and recurrent disease. For nicotine- as well as virus-associated carcinoma, current treatment options besides surgery enclose aggressive radiation and platinum-based chemotherapy regimens. There have been implications for different response rates between HPV-negative and positive carcinoma; however, this has not yet lead to a change in general therapy guidelines. Resistance towards radiation and chemotherapy is a widespread phenomenon in all tumor stages and exists for platinum-based chemotherapy as well as for targeted therapy (Anti-EGFR; epidermal growth factor-receptor) and recently emerging checkpoint inhibition¹⁵. Ineffective radiation and chemotherapy come at a high cost of significant patient morbidity in terms of dysphagia, mucositis, dry mouth and risk of decrease of renal or cardiac function among others. Predicting therapy response prior the decision of a general therapy concept for each individual patient seems to be the crucial goal, preventing unnecessary treatment concepts, side effects and costs.

We sought to establish a model to test individual patient's treatment susceptibility towards current standard chemo-radiation that could be integrated into the regular and quality-controlled oncologic treatment algorithm from a technical standing point. The far goal was to use the model without using heavily altered and aged cell lines, as they poorly represent actual human tumor cells without their variability and heterogeneity as we know now, while establishment of the protocol was done in various cell lines. To be independent only from commercially available cell lines, we recently successfully generated an intermediate cell line called "PiCa" from primary HNSCC cells from human tumor specimens with conserved cellular markers on its surface and limited passages¹⁶. This PiCa cell line should serve as a preparation for the development of the model on the road to then later following trials with fresh human cancer cells from tumor biopsies. It has been shown that cells in three-dimensional cell cultures react differently and more in vivo-like to administration of cancer drugs than those growing in monolayers^{17,18,19,20,21}, mainly due to conservation of migratory and sub-differentiation properties of certain cell subsets^22,23,24. Here, we describe the protocol of a spheroid-based, three-dimensional model from intermediate cell lines and primary human squamous cell carcinoma cells and ways how integrate such a model into cancer treatment of the head and neck surgeon and oncologist (Figure 1).

Protokół

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 sevoflurane, muscle relaxing agent) or local infiltration (2% ultracaine, adrenalin)/surface (xylocaine) anesthesia in the operating room or otolaryngology examination chair. Visualize the tumor mass in oral cavity/pharynx/larynx/other parts of the upper digestive and respiratory tract with standard operating instruments and, if needed, under the microscope.
2. Take a fresh tumor biopsy from the periphery of the cancerous lesion with blunt or cutting instruments. Avoid the center of the lesion due to abundant necrosis in this area. Put the obtained tissue to a sterile container with isotonic sodium chloride solution.
3. After the biopsy, perform hemostasis as needed, e.g., with a bipolar or monopolar coagulation device in addition to the use of vasoconstrictive substances.
4. Bring the tumor biopsy intended to be used for cell culture experiments directly to the adjacent laboratory tract. Send other tumor biopsies to the pathologist as usual to rule out cancer.
5. Make sure that a laboratory technician is ready to process the specimen directly.

2. Processing the Tumor Specimen

1. Place the tumor specimen on a suitable and sterile surface and cut it thoroughly with a sterile single-use scalpel into as little as possible pieces.
   Caution: Make sure the status of infectious and blood-borne diseases is well documented and the technician is in attention of the institutions standard protocols to prevent needle stick or cutting injury with potentially biohazard patient material and the protocols to follow after possible injury.
2. After sufficient mechanical separation of the primary tissue, put the tissue into a vial containing Collagenase I/II and incubate for 1 h at 37 °C. Sieve through a 70 µm falcon cell strainer and wash the suspension with Hanks' Balanced Salt solution (HBSS).
3. After successful separation and subsequent washing, place the suspension containing 1-2 × 10⁶ cells into a T75 cell culture flasks (75 cm²) to grow to sub-confluency at 5% CO₂ and temperature of 37 °C. This step may take up to 10 days.
   1. Use special keratinocyte culture medium consisting of the following: 125 mL of Dulbecco's modified eagles medium (DMEM), 250 mL of Keratinocyte complemented medium (complemented medium consisting of 500 mL of Keratinocyte SF Medium, 15 mg of bovine pituitary extract (BPE), 2.5 mL of penicillin/streptomycin, 150 ng recombinant human epithelial growth factor (EGF), 516 µL of 300 mM CaCl₂, stock mix to be prepared in advance), 125 mL of F12 nutrient mix, 10 mg of BPE (0.75 mL), 75 ng recombinant human EGF (2 µL), 3.75 mL of 200 mM L-Alanyl-L-Glutamin-Dipeptide (Table of materials).

3. Seeding the Cells into Ultra-low Adhesion Cell Culture Plates

1. Confirm tumor cell growth under a microscope. Count the cells in culture (primary or cell culture) and seed 5,000 primary tumor cells or 1,000-2,000 cells of intermediate cell line/other cell line in 200-300 µL of media (step 3.2.) into an ultra-low adhesion plate with concave, round bottoms (96-well).
2. Culture the cells at 5% CO₂ at 37 °C and in equal parts DMEM and airway epithelial cell medium (BEGM), 10% fetal bovine serum, 1% penicillin/streptomycin, 1% sodium pyruvate, 1% non-essential amino acids, 1% L-glutamine (step 2.3.). If cell lines are being used, media on the basis of DMEM is sufficient.
   1. Perform media changes every other day. Pay attention not to aspirate the spheroid with the pipette during media changes. Culture the spheroids until the level of growth as descibed in 3.3 is reached (approximately 7-10 days).
3. Confirm spontaneous spheroid formation under the microscope by looking for three-dimensional, spheroid-shaped cell conglomerates. Exclude the wells with irregular and/or multiple spheroid formation from further investigation.
   NOTE: Spheroids should be visible with the naked eye, too, facilitating further processing and media changes as described below.

4. Exposing Spheroids to Multimodal Standard or Experimental Tumor Therapy

1. Choose a desired therapy regimen. Design sufficiently large control groups that allow comparisons of treatments to untreated spheroids or spheroids receiving only partial therapy regimens, e.g. radiation alone. The size of control groups depends on the experimental group design and cannot be defined universally.
2. Exchange the media to media with additives, meaning media with chemotherapeutics and/or monoclonal antibodies at desired concentrations. Add Cisplatin at the concentrations of 2.5/5/10 µM or 5-fluoruracil (5FU) at 30 µM.
   NOTE: For high throughput experiments or large group sizes/large number of groups, one can use an automated pipetting robot as we are further establishing in the experiments.
3. Alternatively, radiate the cell culture plates with spheroids at 2 Gy using a suitable radiation facility.
   Caution: Respect the institution's protocols regarding prevention of harmful radiation exposure of employees. Work only with designated and trained technicians according to radiation protection guidelines. If desired, add chemotherapeutics as described under 4.2. afterwards.
4. Incubate the cells for 24 h in previously described culture conditions (37 °C, step 3.2).
5. After the incubation, continue the cell culture for at least 6 days with media changes every other day.

5. Assessment of Spheroid Size and Extent of Cellular Proliferation for Assay Read-out

1. Measure the spheroid size in terms of area after digital photo documentation on day 6 (day 10, day 16) with a graphic software (parameter 1).
2. After centrifugation at 520 x g for 2.5 min of the plate, remove the supernatant. Wash the cells with sufficient amount of 1x PBS and centrifuge the plate again as described, followed by removing the supernatant (PBS).
3. Add 100 µL of enzymatic cell detachment solution to each vial to allow the spheroids to dissolve. Incubate the plate for 8 min at 37 °C.
4. Check for successful dissolving of the spheroid under the microscope. Add 100 µL of DMEM. Centrifuge the plate at 520 x g for 2.5 min. Remove the supernatant and suspend the cells in 100 µL of DMEM.
5. Perform a commercially available colorimetric proliferation assay, in example WST-8 assay on each vial according to the manufacturer's instructions²⁵. Read out the assay in an enzyme-linked immunosorbent assay (ELISA) reader (parameter 2).

Wyniki

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 ...

Dyskusje

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...

Materiały

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