Hyaluronic acid-based hydrogels as 3D matrices for in vitro evaluation of chemotherapeutic drugs using poorly adherent prostate cancer cells

Abstract

The current investigation aimed to develop a biomimetic, three-dimensional (3D) culture system for poorly adherent bone metastatic prostate cancer cells (C4-2B) for use as an in vitro platform for anti-cancer drug screening. To this end, hyaluronic acid (HA) derivatives carrying complementary aldehyde (HAALD) and hydrazide (HAADH) groups were synthesized and characterized. In situ encapsulation of C4-2B cells was achieved by simple mixing of HAALD and HAADH in the presence of the cells. Unlike two-dimensional (2D) monolayer culture in which cells adopt an atypical spread morphology, cells residing in the HA matrix formed distinct clustered structures which grew and merged, reminiscent of real tumors. Anti-cancer drugs added to the media surrounding the cell/gel construct diffused into the gel and killed the embedded cells. The HA hydrogel system was used successfully to test the efficacy of anti-cancer drugs including camptothecin, docetaxel, and rapamycin, alone and in combination, including specificity, dose and time responses. Responses of cells to anti-neoplastics differed between the 3D HA hydrogel and 2D monolayer systems. We suggest that the data obtained from 3D HA systems is superior to that from conventional 2D monolayers as the 3D system better reflects the bone metastatic microenvironment of the cancer cells.

Figure 1. Chemical structures of materials used in HA hydrogels

Covalent cross-linking of HAALD and HAADH in PBS produces in a stable hydrogel via the formation of hydrazone bonds, with water being the only by-product.

Figure 2. C4-2B cell viability is retained when cultured in the HA hydrogels

Phase microscope images of cell cluster growth in HA hydrogel over seven days (A). Phase microscope images of cell growth on 2D plastic monolayer over seven days (B). Viable cell numbers in HA hydrogel compared to plastic over seven days (C). Trypan blue positive dead cells were excluded from these counts. Basal level of cell death in HA hydrogel or on plastic over seven days (D). Dead cells were counted after staining with trypan blue. * p<0.05, n = 3, error bars = SEM.

Figure 3. C4-2B cells form clusters in HA hydrogels but not on plastic

Phase contrast images of cells cultured on plastic (A) or in HA hydrogel (B) for 2 days. Confocal images of live/dead stained cells cultured on plastic (C) or HA hydrogel (D) for 2 days. Cells were stained with Syto-13 (green) for live cells and PI (red) for dead cells. Confocal images of cells cultured on plastic (E) or HA hydrogel (F) for 2 days stained for F-actin with phalloidin (green) and Draq5 for nuclei (blue).

Figure 4. Camptothecin-treated C4-2B cells cultured in HA hydrogel die from the outside in

Live/dead staining of cells in HA hydrogel treated with DMSO as a vehicle control (A) or treated with 0.574 nM CPT for 24 hours (B). Expanded horizontal and vertical views of a single cluster of cells in HA hydrogel treated with 0.574 nM CPT for 24 hours (C). Live/dead staining as in Figure 2 shows significant cell death (red) beginning on the periphery of the clusters.

Figure 5. Camptothecin and Docetaxel diffuse from the medium into the HA hydrogel

Efficiency of diffusion of CPT from the medium into the HA hydrogel after 24 hours (A). Efficiency of diffusion of DOX from the medium into the HA hydrogel after 24 hours (B). Error bars = SEM, n = 3.

Figure 6. C4-2B cells are most sensitive to camptothecin when administered as a single agent

Dose response curves for killing by DOX and RAP were compared to that for CPT in HA hydrogel or on plastic. Drug induced killing by CPT after a 24 hour treatment; n = 4 (A). Apoptotic response to DOX after 48 hour treatment; n =2 (B). Apoptotic response to RAP after 48 hour treatment; n=2 (C). Error bars = SEM, * p < 0.05 comparing HA hydrogel and plastic cultures.

Figure 7. Apoptosis in C4-2B cells is higher in HA hydrogel than plastic in response to single or combination drug treatment

Levels of apoptosis over a 24 hour time course with 1.435 nM CPT (A). Levels of apoptosis after 24 hours with 0.574 nM CPT and 2 nM DOX (B). Levels of apoptosis seen after 48 hours with 2 nM DOX and 20 nM RAP (C). Error Bars = SEM, n = 3, * p < 0.05.