Sponge-supported cultures of primary head and neck tumors for an optimized preclinical model

Abstract

Treatment of advanced head and neck cancer is associated with low survival, high toxicity and a widely divergent individual response. The sponge-gel-supported histoculture model was previously developed to serve as a preclinical model for predicting individual treatment responses. We aimed to optimize the sponge-gel-supported histoculture model and provide more insight in cell specific behaviour by evaluating the tumor and its microenvironment using immunohistochemistry. We collected fresh tumor biopsies from 72 untreated patients and cultured them for 7 days. Biopsies from 57 patients (79%) were successfully cultured and 1451 tumor fragments (95.4%) were evaluated. Fragments were scored for percentage of tumor, tumor viability and proliferation, EGF-receptor expression and presence of T-cells and macrophages. Median tumor percentage increased from 53% at day 0 to 80% at day 7. Viability and proliferation decreased after 7 days, from 90% to 30% and from 30% to 10%, respectively. Addition of EGF, folic acid and hydrocortisone can lead to improved viability and proliferation, however this was not systematically observed. No patient subgroup could be identified with higher culture success rates. Immune cells were still present at day 7, illustrating that the tumor microenvironment is sustained. EGF supplementation did not increase viability and proliferation in patients overexpressing EGF-Receptor.

Keywords: cell culture; head and neck cancer; primary tissue; sponge gel supported histoculture; squamous cell carcinoma.

Figure 1. Culture efficacy data from all single tumor fragments (dots) at day 0 and day 7 cultured with standard RPMI

Depicted are the raw and normalized values for each tumor fragment. Values shown in the graph correspond to the horizontal bar which depicts the median from all included fragments.

Figure 2. The effect of the culture optimization conditions on tumor viability and proliferation in comparison to standard RPMI, at day 7

(A) Boxplot of normalized viability and proliferation (median and range) of all tumor fragments cultured with the various optimization conditions. The horizontal red line delineates the median value for the standard RPMI. (^*Best conditions.) (B) Scatter plot of raw median viability and proliferation percentages per optimization condition when compared to standard RPMI, depicted per individual patient. A single data point represents the difference between the median individual percentage of an optimization condition and the standard RPMI. The red bar indicates the median of all these single data points within that specific condition. (^*Best conditions.) (C) Scatter plot of normalized data per patient for the three best selected optimization conditions (EGF 50 ng/ml ◊, Hydrocortisone 0.4 μg/ml □ and Folic Acid 6 mg/L ▼) versus standard RPMI. One symbol resembles the median of all fragments per individual patient; error bars around the symbols range from the first to the third quartiles. The size of the symbol is inversely proportional to the p-value of a two-sided Mann–Whitney U test comparing the RPMI and the tested optimization condition within one patient. The green circles indicate the selected samples for EGFR and immune cell IHC. For comparison between figures and tables, red numbers indicate individual patients. (Mind the axes that vary between the graphs.)

Figure 3. The presence of immune cells during histoculture

(A) Visualization of CD3, CD8 and FoxP3 T-cell staining at day 7. In the merged image, one can distinguish between T helper cells (CD3+), cytotoxic T cells (CD3+, CD8+) and regulatory T cells (CD3+, FoxP3+). (B) Visualization of CD68 and CD163 macrophage staining at day 0. In the merged image, one can distinguish M2 macrophages (CD68/CD163).

Figure 3. The presence of immune cells during histoculture

(A) Visualization of CD3, CD8 and FoxP3 T-cell staining at day 7. In the merged image, one can distinguish between T helper cells (CD3+), cytotoxic T cells (CD3+, CD8+) and regulatory T cells (CD3+, FoxP3+). (B) Visualization of CD68 and CD163 macrophage staining at day 0. In the merged image, one can distinguish M2 macrophages (CD68/CD163).

Figure 4. Illustration of the sponge-gel-supported histoculture method and immunohistochemistry read-out

(A) Biopsies from previously untreated HNSCC patients are taken under general anaesthesia after informed consent. Biopsies are transported to the laboratory within 1 hour and cut into single fragments. Each single fragment is cultured on a sponge drenched into medium in a 12-well plate. The fragment is placed on the sponge in such a way that it is surrounded by air and attached to the sponge enabling it to absorb medium. Using this method, the in-vivo situation is simulated. (B) Illustration of the pathological scoring system. Of each single tumor fragment, at day 0 and 7, three slides are cut and stained for pan-Cytokeratin, Hematoxylin and Eosin (H&E) and Ki-67. With the H&E and pan-Cytokeratin staining the percentage of tumor is scored (% Tumor, cancer cells). With the H&E staining the percentage of viable cancer cells in relation to the total amount of tumor (including areas of necrosis) is determined (% Viability). The Ki-67 staining is used to determine the proliferation rate of the viable cancer cells (% Proliferation).