Patient-Derived Spheroid Culture Models Are Better Than Monolayer Models in Chondrosarcoma Research

Purpose Chondrosarcoma (CSA) are mesenchymal tissue-derived bone tumors. CSA mainly occurs in older people. CSA has demonstrated resistance to chemotherapy and radiation; complete surgical removal with negative margins is the only treatment option. In the case of metastatic CSA, the chance of survival is meager. Since the conventional two-dimensional cell culture models failed to retain tumor characteristics, developing preclinical models mimicking the disease with the highest fidelity is paramount for personalized treatments. Methods In this study, we established spherical cultured cells as new models for CSA. First, we demonstrated that CSA cells could form spheroids when cultured in ultra-low attachment plates. Next, tissue samples from CSA patients were collected and processed into primary cells, which were subsequently cultured as primary spheroids. The growth rate of primary spheroids was monitored and the histology of mature spheroids were characterized. These primary spheroids were used in drug susceptibility studies where traditional doxorubicin therapy and our novel disulfiram-copper therapy were tested. Results Compared with conventional monolayer cultures, spheroids better recapitulated the features of the in vivo tumor in the aspect of the formation of extracellular matrix. In the drug susceptibility study, spheroids demonstrated high resistance to the classic therapies, suggesting that monolayer cultures may give false positive results. Therefore, using spheroids for drug research and development in the CSA field should provide more accurate results. Conclusion In summary, our study of primary CSA spheroids brought new insight into their chemoresistance and demonstrated its potential for personalized treatment of CSA in clinical medicine.


Introduction
Chondrosarcoma (CSA) is a collective term of a group of heterogeneous malignant bone tumors which are normally characterized by common production of hyaline cartilaginous matrix.CSA arise mostly in adults and is the second most common bone malignancy after osteosarcoma, accounting for approximately 20% of all bone malignancies, and it has been reported about 3 new cases per million each year 1 .CSA is classi ed on a continuum of histologic grades, from low grade CSA to dedifferentiated CSA, and the incidence of metastases and prognosis are related to tumor grade [2][3][4] .CSA is a so-called surgical disease, which means until now complete surgical removal of the lesion before metastases is the only way to cure [5][6][7] .Therefore, it is necessary to furtherly understand the mechanism of CSA development and to develop novel treatment for those unresectable cases.However, one of the greatest barriers to CSA basic research is the lack of reliable models.CSA are usually abundant in extracellular matrix (ECM), and the conventional monolayer cultures fail to retain the complexities of tumor microenvironment of CSA 8,9 .This lead to the development of three dimensional cultures, including spheroids, organoids and scaffold-based tissues [10][11][12] .Among these options, spheroids are the most straightforward, and the protocol of culturing is easily standardized.Several studies have demonstrated the feasibility of CSA spheroids culture and advantages of the model in aspect of recapitulating tumor oxygen gradients, ECM formation and high resistance in cytotoxicity assay [13][14][15] .However, whether there is difference between low grade CSA spheroids and dedifferentiated CSA spheroids remain to be investigated.We believe this would help to understand both CSA itself and the spheroids models.
In this study, we asked: (1) Are there differences between low grade and dedifferentiated CSA spheroids in aspects of morphology, histology and chemoresistance?(2) Do both types of patient derived CSA spheroids recapitulated tumor morphology and chemoresistance?We hypothesize that both low grade and dedifferentiated patient derived spheroids retain the own characteristics of their original tumors and thus, patient derived spheroids are ideal models for CSA basic research.

Methods
Patient samples and primary cell culture Institutional review board approval (PRO10050461) was obtained from UPMC Shadyside Hospital for this study.Informed consent to participate in our tumor registry and tissue bank were obtained to collect samples from patients.Once the sarcoma has been removed from the operative eld, the surgeon performs the intralesional removal of sarcoma tissue and the tumor was transported to the lab.Tumors were minced and dissociated into single-cell suspensions using the MAC human tissue dissociation kit (Miltenyi Biotec, Auburn, CA, USA).Primary cells were cultured in asks (37° C, 5% CO2) with DMEM (Corning, Manassas, VA, USA).Medium was changed every three days or when medium turned yellow.
Another 100 µL of Fresh medium was added to each well after 3 days of seeding (day 3).The medium was changed every 3 days or when medium turned yellow.To change the medium, 100 µL of supernatant was gently aspirated from each well and 100 µL fresh medium was added.All the cultures were maintained in a 5% CO 2 humidi ed environment at 37°C.Spheroid growth was monitored and images were captured using an Olympus IX81-Motorized Inverted Microscope (Olympus, Center Valley, PA, USA).Spheroid size was measured by cellSens Standard software (Olympus Life Science, Waltham, MA, USA).

Agarose embedding and histology
Spheroids were cultured for 10 days, and medium was gently aspirated from each well.Spheroids were washed three times with PBS.Spheroids were xed with 4% paraformaldehyde (Electron Microscopy Sciences, Hat eld, PA, USA) for 30 minutes.After xation, spheroids were transferred to tissue culturing dishes and embedded with 4% agarose.Solidi ed agarose was cut into small cubes with spheroid in the center.Cubes were dehydrated successively in 70% isopropanol for 2 hours, 96% isopropanol for 2 hours, 100% isopropanol overnight and 100% acetone for 2 hours.Dehydrated agarose cubes were incubated in pre-warmed liquid para n for 2 hours before para n embedding.5-µm-thick sections were cut and adhered to poly-L -lysine-coated glass slides.For monolayer culture, single cell suspension was obtained from ask after trypsinization and were seeded into Nunc Lab-Tek II Chamber Slide (Thermo Scienti c, Rochester, NY, USA) at an initial density of 80,000 cells with 2 mL medium per chamber.After adherence to the slides, chambers were removed.Both monolayer culture and spheroids slides were stained with hematoxylin and eosin after depara nization and rehydration.Imaging was performed with the Nikon Eclipse E800 microscope (Nikon Instruments, Melville, NY, USA) at magni cations of 20 x and 40 x.
Images of the tumors; pathologic sections were taken with an Olympus BX45 microscope (Olympus, Center Valley, PA, USA) by a musculoskeletal pathologist in UPMC Shadyside Hospital.

Cytotoxicity assay
For monolayer cultures, 20,000 cells/well were seeded into at bottom 96-well plates (Corning Inc, Kennebunk, ME, USA) with 100 µL of culture medium and left to adhere overnight.Doxorubicin hydrochloride (LC Laboratories, Woburn, MA, USA), disul ram (Sigma-Aldrich, St. Louis, MO, USA) were then added to each well.For 3D culture group, spheroids were seeded in Nunclon Sphera 96-well round bottom Microplates at density of 20,000 cells per well and were maintained for 7 days.Drugs were applied at day 8 of spheroids culture.Doxorubicin was diluted using DMEM, and three-fold dilution from the highest concentration of 100 µM to lowest concentration of 5 nM was achieved.Cells and spheroids were incubated in different concentrations of doxorubicin for 48 hours in a 5% CO2 humidi ed environment at 37° C, and the cell viability was measured.To measure cell viability after drug treatment, 10 µL of PrestoBlue (Invitrogen, Carlsbad, CA, USA) was added to each well and plates were placed in the incubator for 25 minutes for 2D cultures and 3 hours for spheroids.Fluorescence was measured with a Synergy HT Multi-Mode Microplate Reader (BioTek, Winooski, VT, USA) with an excitation and emission wavelength of 560/590 nm.

Statistical analysis
All quantitative data in this study was generated from at least three independent biological replicates.All graphs are presented in form of mean value with standard deviation.Statistical analysis was performed using Graphpad Prism 8 (GraphPad, La Jolla, CA, USA).*p < 0.05, **p < 0.005, ***p < 0.001, ****p < 0.0001.

Generating and Growth Monitoring of Low Grade CSA Spheroids
The spheroids generated from both cell line and low grade CSA patient samples could be maintained for over 10 days.After SW1353 was rst seeded into ULA plates and incubated overnight, the SW1353 spheroid formed at day 1, with diameter of 354.17 ± 6.03 µm.The size of spheroids decreased from day 1 to day 7 (166.67± 5.44 µm).At day 10, the diameter was 158.33 ± 3.75 µm.For CS15, spheroids formed at day 1 with diameter of 483.33 ± 7.47 µm.After formation, spheroid shrunk and became denser in the core area.The diameter of day 10 spheroid was at 279.17 ± 5.62 µm.S564 cells grew in the same pattern as CS15 did.The diameter of day 1 S564 spheroid was at 688.24 ± 9.42 µm, and it decreased to 300.00 ± 5.81 µm at day 10.As red arrow shows in Fig. 1, we observed transparent matrix around S564 spheroid at day 7 and day 10, which did not exist at earlier stage (Fig. 1).The S26 spheroids were observed with the largest diameter of 770.83 ± 10.21 µm, compared with that of other samples at day 1.We observed the same growth pattern of S26 as that of other samples.The well-formed spherical shape was observed at day 7, and the diameter was of 508.33 ± 4.77 µm.At day 10 of culture, the S26 spheroids began to collapse and the main body of the spheroids were surrounded by lots of cell debris (Fig. 1).

Generating and Growth Monitoring of Dedifferentiated CSA Spheroids
HT1080 spheroids formed at day 1 with a diameter of 729.17 ± 10.56 µm, and increased in cell quantity but decreased in size (645.83± 6.27 µm) at day 3.The spheroid grew into a mature round shape and then proliferated and expanded from day 3.It reached diameter of 854.17 ± 11.77 µm at day 10, and the 2 layers structure that dark core and light peripheral area was observed under microscope.We observed two different growth patterns of dedifferentiated CSA spheroids.The rst group included CS23, S115 and S166 spheroids which the shrunken growth patterns were observed.The size of day 1 CS23 spheroid was 708.33 ± 9.73 µm, and 729.17 ± 8.97 µm, 604.17 ± 7.52 µm for S115 and S166 respectively.At day 3, we observed the density of these 3 spheroids was signi cantly increased.From day 3 to day 10, the sizes kept decreasing and reached 301.67 ± 5.66 µm, 479.17 ± 4.43 µm, 301.67 ± 4.01 µm for CS23, S115, S166 correspondingly.At day 10, cell debris can be observed around S166 spheroids.The second group consisted of CS21b and S494.After formation of spheroids, CS21b and S494 spheroids enlarged from diameter of 750.00 ± 6.98 µm and 695.65 ± 8.84 µm at day 1, to diameter of 958.33 ± 14.62 µm and 739.13 ± 10.66 µm at day 10 correspondingly (Fig. 2).
Size of Spheroids Differed from Low Grade CSA Group to Dedifferentiated CSA Group Based on our bright eld microscopy images of two pathological groups of low grade CSA and dedifferentiated CSA spheroids, we found the average size of dedifferentiated CSA spheroids were larger than that of low grade CSA spheroids, though we seeded the same amount of cells into each well.The signi cant difference (p = 0.029) in spheroid size (288.54± 91.64 µm v.s.605.69 ± 91.64 µm) was observed in day 10 (Fig. 3).

Histology of primary cells, spheroids and original low grade tumors
In the S26 tumor, abundant hyaline matrix was observed with an approximate cellularity at 40% − 60%.The nuclei of cells were round and occasionally spindled.Two mitosis per 10 high power eld (hfp) was recorded.In S26 spheroid, some eosinophilic matrix was observed and the cellularity was scored between 40% -60%.Nuclei of cells were spindled and occasionally degenerated.Clear boundaries were formed by spindled cells at the periphery.The size of monolayer cells was large, and con uence was at 95%.For CS15, small, round oval cells were observed in the tumor and the cellularity was of 20% − 40%.Stellate and hyperchromatic nuclei were observed.The ECM was abundant.The corresponding spheroid was formed by pleomorphic spindle cells with cellularity at 40% − 60%.ECM was observed inside.Some cells were degenerated.The cytoplasm was granular in the spheroid.The boundary was well formed.The monolayer cells were at 100% con uence and mitoses were observed.In S564 tumor, vacuolated cells were observed.The cellularity of the tumor was at 40%-60%.Hyaline matrix was formed and the eosinophilic component could also be observed.In the spheroid, the cellularity was of 60% − 80%.Both spindled cells and round cells were observed and pleomorphic was presented.Vacuolation was found inside the spheroid.The boundary was well maintained by cells in periphery.For monolayer cells, they were lightly stained and were at con uence of 40% (Fig. 4).

Histology of primary cells, spheroids and original dedifferentiated tumors
Dedifferentiated CSA tumors demonstrated similarities that they all had high cellularity and presented low ECM formation.For CS21b, pleomorphic and spindle cells were observed in the tumor.Neutrophils and lymphocytes in ltrated.The mitotic activities were at 60 per 10 hpf.For corresponding spheroid staining, the density of cells was increased at the edge and decreased in the core area.In the middle of the spheroids, cells were observed more spindle shaped and degenerated cells could also be found.Round cells were observed mainly at peripheral area, and there were detached individual cells at the area.The CS21b 2D cells were at about 90% con uence, and the nuclei were deeply stained.Mitosis was observed in the monolayer cultures.For CS23, the tumor was of about 100% cellularity with a few lymphocytes in ltrating.Cells were pleomorphic and spindled.Mitotic activity was about 115 cells per 10 hpf.In spheroid, cellularity was at about 60% − 80%.Cells were vacuolated in the middle and degenerated cells were observed.Cells at the periphery were spindled and a clear capsule-like boundary formed.There were no cells detached from the spheroid.For monolayer cells, they were at 50% con uence and were lightly stained by H&E.For S115, there was no ECM formation in the tumor and cellularity was at 60% − 80%.Cells were pleomorphic and some rhabdoid cells presented.Lymphocyte in ltration was also observed.Mitosis was found about 15 cells per 10 hpf.ECM was not formed and cellularity was about 80% − 100%.Pleomorphic and vacuolated cells were located in the middle.The cells at the outer layer were deeply stained and were all spindled, therefore a capsule-like boundary formed, and no cell was detaching.S166 tumor was composed of pleomorphic oval cells.Both round and spindled nuclei were observed.The cytoplasm was amphophilic.Neutrophils and lymphocytes in ltrated.26 mitotic cells were observed per 10 hpf.The cellularity of the tumor was about 60% − 80%.In the corresponding spheroid, most of cells were round and a few were spindled.Degenerated cells also existed in the spheroids.The S166 monolayer cells were imaged at about 60% con uence.In S494 tumor, lots of lymphocytes in ltrated and cells were observed in round shapes with pleomorphic nuclei.ECM was not found in the tumor and cellularity was about 80% − 100%.The mitotic activity was high as 70 cells per 10 hpf.In the spheroids, cells were at high density at the peripheral area compared with cells in the core.Whereas lots of cells were loosely attached and detached cells were also observed.The cellularity of the spheroid was about 80% − 100%.Cells were round with pleomorphic nuclei.The con uence of corresponding monolayer cells was about 30% (Fig. 5).

Spheroids Recapitulate Tumors Better in Morphology Compared with Monolayer Cultures
We compared scores of cellularity of low grade tumors to that of dedifferentiated tumors.The mean value of the low grade group was 2.667 and the mean value of the dedifferentiated group was 4.600.Signi cant differences were demonstrated between the two groups of tumors (p = 0.003).Comparing spheroids between two different groups, the cellularity score of dedifferentiated spheroids (mean value = 4.400) was signi cantly (p = 0.040) higher than that of low grade spheroids (mean value = 3.333).we also compared scores of samples in the same group.The cellularity of monolayer cultures was at 100% in both groups.When comparing between monolayer cultures, spheroids and tumors in dedifferentiated groups, no signi cant difference was observed.In the low grade CSA group, we observed a signi cant decrease of score in the spheroids (p = 0.0088) and tumors (p = 0.0016) when compared to the monolayer cultures.Furthermore, there was no marked differences in cellularity between the low grade CSA spheroids and tumors (Fig. 6).

Spheroids Have Higher Resistance towards Doxorubicin Treatment Compared with Monolayer Cultures
2 CSA cell lines (HT1080 and SW1353) and 6 CSA primary cells (S166, S494, CS15, CS21b, CS23, S564) were cultured as both monolayer cells and spheroids.Cultures were treated by different doses of doxorubicin.For SW1353, IC50 of 2D cultures were at 70.8 nM and IC50 of spheroids were at 0.827 µM (Fig. 7a).The IC50 of HT1080 monolayer cells and spheroids were 0.875 µM and 14.56 µM respectively (Fig. 7b).We next performed the assay on our patient-derived spheroid models.S166 was the only one that we did not observe signi cant differences between 2D and 3D cultures.The IC50 of S166 2D and 3D cultures was 2.54 µM and 3.26 µM respectively (Fig. 7c).Large differences were observed between S494 monolayer cells and spheroids, the IC50 of which was 0.255 µM and 14.7 µM correspondingly (Fig. 7d).In CS21b, signi cant differences in IC50 was also observed between two models, with 7.26 µM of monolayer cultures and 121.9 µM of spheroids (Fig. 7e).CS23 spheroids (IC50 = 8.09 µM) were more resistant to doxorubicin compared with monolayer cells (IC50 = 2.96 µM) (Fig. 7f).The same pattern was also observed in CS15 and S564 samples, the spheroids of which were more resistant to corresponding monolayer cultures.The IC50 of CS15 2D cells was 71.9 nM and of 3D cells was 8.70 µM (Fig. 7g).The IC50 of S564 monolayer cells was 0,723 µM and 0f spheroids was 3.30 µM (Fig. 7h).We summarized the R-square values of these curves, corresponding LogIC50, as well as p values of comparison of ts in Table 1.The mean value of LogIC50 of low grade monolayer cells were − 0.81 ± 0.47, and mean value of dedifferentiated monolayer cells were 0.22 ± 0.50.the signi cant difference was observed between these two group of 2D cells that dedifferentiated monolayer cells were more resistant to doxorubicin treatment (p = 0.047).When comparing differences between two groups of spheroids, dedifferentiated spheroids had a higher mean value of LogIC50 at 1.17 ± 0.52, and that of low grade spheroids was at 0.46 ± 0.42, however, the difference was not signi cant (p = 0.13).We then compared monolayer cultures to spheroids, in both group, a signi cant difference (p (low grade) = 0.047; p (dedifferentiated) = 0.030) was found, and spheroids were more resistant to doxorubicin chemotherapy (Fig. 8a).We then tted the curve by linear regression The trend was observed that those monolayer cells with higher resistance tended to be less sensitive to doxorubicin when they were cultured as spheroids and there was a weak correlation with R-square of 0.306 and p value of 0.155.(Fig. 8b).There was a strong correlation that those spheroids, which had a larger dimeter when treated with doxorubicin, were more resistant to the treatment.We performed the linear regression and got an R-square of 0.77, and the p value of 0.0067, which indicated the resistance was strongly related to the size (Fig. 8c)

Discussion
In this study, we successfully generated spheroids not only from HT1080 and SW1353 cell lines, but also from 8 different patient derived primary cells.The results demonstrated the feasibility of using ULA plates for 3D spheroids culture of primary CSA cells.Besides, most of the formed spheroids can be maintained more than 10 days, then cell debris around the spheroids could be observed which indicated necrosis happened.However, there was still study indicating spheroids could be maintained for a longer time 14,16 .This might be related to cell types.By using the ultra-low attachment plates, we were able to grow spheroids as uniformed shapes and sizes, which were essential for repeatability of further studies, i.e. histology, RNA sequencing, cytotoxicity assay, etc.
We divided cells into two groups based on their original pathology, low grade CSA and dedifferentiated CSA.Two types of growth patterns were observed, which were the contraction patterns and the expansion patterns.The phenomenon was also reported by others 17 .All low grade spheroids shrunk during culture, while some of the dedifferentiated spheroids shrunk, and the other expanded.More interestingly, S494 is the recurrence of S166, however, the S494 spheroids expanded in size from day 3 to day 10, while S166 spheroids presented the shrunken pattern over time.The possible explanation were: 1) the growth pattern was decided by proliferating ability of the cells.The cells from low grade chondrosarcoma had lower proliferation rate and invasion ability, leading to the contraction of spheroids.This could also explain the different patterns of S166 and S494 spheroids, as recurrence was usually more malignant than the original counterpart.2) the cells originated from low grade cartilaginous component of dedifferentiated CSA tended to grow in a contraction pattern, and those from dedifferentiated component tended to expand.This was reasonable, because for each dedifferentiated CSA, both components were presented in the tumor, and the behaviors of these two components were different 18,19 .When we dissociated the tumor, we were not sure which cells were included or dominant in spheroids.Some studies also claimed that the growth pattern of spheroids were regulated by β1 integrin 20 and E-cadherin 21,22 .The β1 integrin was important for cartilage development and mediated cell-matrix interaction, which diminished cell migration in low grade CSA 23,24 .Therefore, this might result in contraction pattern of low grade CSA spheroids.For E-cadherin, it was only expressed in certain kinds of soft tissue sarcomas but not chondrosarcoma 25 .
Then, we mainly made comparisons of histology among monolayer cultures, spheroids and their original tumor samples.In both low grade and dedifferentiated groups, spheroids recapitulated tumor morphology better than monolayer cultures did in aspects of cellularity, ECM and cell distribution, because spheroids demonstrated higher ECM formation and more speci c spatial distribution, compared with monolayers.However, in some low grade CSA groups, although spheroids were making ECM, the staining of the matrix was not the same as that of the hyaline matrix that existed in tumors.In our humble opinion, spheroids were not producing the specialized hyaline matrix, but some more fundamental component, e.g.collagen II 14,15 .
In the cytotoxicity assay, we demonstrated that in all primary samples, except S166, spheroids were highly resistant to doxorubicin treatment, compared with corresponding monolayer cultures.This was also reported in a study of CSA 3D cell pellets, which were similar to spheroids 26 .This indicated we might obtain false positive results if we only use monolayer cultures as models in cytotoxicity assay, which may partially explain why lots of clinical trials turned out to be disappointing from bench-side to bed-side.The chemoresistance of spheroids were related to their size that larger spheroids were more resistant to doxorubicin.In our opinion, the larger size meant greater di culty for drug molecules to penetrate the spheroids and this was also observed in another study 27 .Besides, when spheroids expanded, cells in the core area would get less oxygen and nutrients, as was reported that hypoxia is also a factor contributing to chemoresistance 28 .Although the sensitivity to doxorubicin differed from one sample to another, dedifferentiated samples were relatively more resistance to doxorubicin in both forms of monolayers and spheroids compared to low grade counterparts, and this demonstrated spheroids could retain the characteristics of their original tumors in aspect of chemoresistance, as dedifferentiated CSA was generally more resistant to chemotherapies than low grade CSA 29 .
this study was the rst to compare differences between low grade CSA and dedifferentiated CSA spheroids.And it was also the rst to reveal the differences and similarities among monolayer cultures, spheroids and their original tumors in aspect of histology.There were some limitations of this study: 1) due to the low incidence of CSA, we were not able to get a larger sample sizes during this study.However, we would continue collecting samples and performing assays on a larger scale in the future.2) the histology part was semi-quantitative and it was inevitable for us to evaluate the spheroid models subjectively.To minimize the impact of personal subjective will, the evaluation of histology features, i.e. cellularity, matrix formation, cell morphology, etc., was conducted by a pathologist from UPMC Shadyside Hospital, who was not familiar with the aim of the project, and slides were also blinded before being sent for scoring.3) we did not furtherly investigate molecular mechanisms under our ndings, so in the future, we would focus on mechanism studies and try to explore more aspects of patient derived CSA spheroids, for instance, performing the bulk RNA-sequencing of all our samples to understand the relationship between our models and original tumors in genetic and molecular aspects.

Conclusions
In conclusion, both patient derived low grade and dedifferentiated CSA spheroids recapitulated their own tumor characteristics in aspect of morphology and chemoresistance.Spheroids were novel and ideal

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