Cancer Therapy Vol 3, 359-364, 2005

Selective tumor cell growth on tissue culture polystyrene and Primaria^TM: a preliminary study

Research Article

Shamim A. Faruqi^1,*, Okeychukwu A. Ibeanu¹, Tariq Ali¹, Harvey b. Spector², Joel S. Noumoff¹

¹Department of OB/GYN

²Department of Histopathology, Crozer-Chester Medical Center, One Medical Center Boulevard, Upland, PA 19013.

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*Correspondence: Shamim A. Faruqi, Ph.D., Gynecologic Oncology Research Laboratory, Department of OB/GYN, Crozer-Chester Medical Center, Upland, PA 19013, USA; Tel: 610-447-2775; Fax: 610-447-2939; e-mail: gynoncob@aol.com

Key words: tumor cell growth, polystyrene and Primaria^TM, tissue culture

Abbreviations: normal tissue culture plastic, (NTCP)

Received: 15 March 2005; Revised: 10 June 2005

Accepted: 10 June 2005; electronically published: June 2005

Summary

We have grown endometrial and ovarian tumors of different grades separately, either on normal tissue culture plastic (NTCP) or Primaria^TM and compared their growth characteristics. These preliminary results have shown that well differentiated and moderately differentiated tumors of both ovarian and endometrial origins grow better on Primaria^TM than NTCP when compared to poorly differentiated tumors. These results may have relevance to prognosis, treatment planning and an understanding towards in-vivo behavior of tumor cells. Because low-grade tumors have a strong growth preferential for Primaria^TM, it would be interesting to see the final outcome of those tumors which are medium- or high-grade and grow favorably on it.

I. Introduction

In-vitro culture and growth of tumor cells usually employs normal tissue culture plastic (NTCP), which is essentially polystyrene impregnated with oxygen containing molecules. The high oxygen content makes this substrate more hydrophilic and hence is thought to enhance cell adhesion capacity. Primaria^TM (Beckton Dickinson Labware, Franklin Lakes, NJ, USA), a modified polystyrene tissue culture plastic containing almost equal amounts of nitrogen and oxygen, is contended by the manufacturer’s unpublished data to have a superior cell adhesion quality than NTCP. Earlier, one of the current authors (Fuscaldo et al, 1989; Faruqi and Krueger 2004) cultured lung cancer cells both on NTCP and Primaria^TM and discovered that tumor clones had a significant growth differential with regard to these two substrates. Certain clones which were absent on one substrate were present in the other (and vice versa).

Similarly, genomic differences were also discovered in the ovarian and endometrial tumor cell cultures between these two substrates (Faruqi et al, 2003). From this laboratory, we further reported data on 35 ovarian tumors of different grades and noted that when a certain tumor failed to grow on one substrate, it successfully grew on the other, increasing the rate of positive tumor culture (Deger et al, 1997).

In the present report, we point out a relationship between successful cell culture in each of the tumor grades and the two substrates separately, both in endometrial and ovarian malignancies.

II. Materials and Methods

This study included twenty-one epithelial endometrial and twenty-five epithelial ovarian primary tumors, classified as either well, moderately, or poorly differentiated. After accession by the histopathologist, the material was brought to the laboratory in serum-free RPMI-1640. Material was processed and plated as described earlier, through the creation and separation of cell slurries in RPMI and 10% fetal bovine serum (FBS) supplemented with 2% penicillin and streptomycin, with 2mM glutamine (Deger et al., 1997). Two mL of a cell culture containing 5*10⁴ cells/mL were plated (for a plate total of 1*10⁵ cells/plate) and cultured in 60 mm of either NTCP or Primaria^TM Petri-plates at 37^oC with 5% CO₂ at 98% humidity. Three replicates of each cellular sample were made. For in-situ harvest, cells were first exposed to a hypotonic solution (a 1:1 mixture of 0.4% each sodium citrate and potassium chloride) and incubated at 37^oC for 35min. Twenty drops of Carnoy’s fixative were added to the hypotonic solution as a pre-fixative and the plates were incubated at room temperature for 10 min. The solution was then exchanged for fresh fixative two times for 20 min each, and after a final change of fixative, incubated for 10 min. For harvest, Petri-plates were washed twice in fresh fixative and placed to dry under a slow fan in an atmosphere of 78^oC temperature and 36% humidity. Chromosomes were banded after a pretreatment with a highly purified trypsin solution (Enzar-T^TM, 0.5mL in 75mL Hanks’ Balanced Salt Solution 1X, pH adjusted to 7.2 through NaHSO₄), and stained with either Geimsa or Leishman stains. The rims of the Petri-plates were then removed using a pair of pliers. The remaining discs, containing the stained cells, were cut into halves with a hot knife and each half disc was mounted on a glass slide. Entire slides were scanned under a high power compound microscope and all dividing cells were scored. Bar graphs were made from the average score of the three replicate Petri plates.

III. Results

A total of 46 gynecological tumors were cultured, of which 21 were endometrial in origin while 25 came from the ovarian sites. Among the endometrial tumors, 3 were well differentiated (low grade tumors), 6 were moderately differentiated and 12 were poorly differentiated (high grade tumors). Of the ovarian tumors, 10 were well differentiated while 15 were poorly differentiated. There were no moderately differentiated ovarian tumors. Results were analyzed and presented separately according to the primary tumor site and grade (Figures 1, 2).

A. The endometrial tumors

The three well differentiated tumors grew well in Primaria^TM dishes while tumors from patients 2 and 3 displayed no growth on NTCP plates (Figure 1A). In the moderately differentiated tumors, out of six samples three showed more growth on Primaria^TM than on NTCP while one grew exclusively on Primaria^TM and similarly one on NTCP. Only in a single case there was more growth on NTCP than on Primaria^TM in this tumor category (Figure 1B). Of the poorly differentiated tumors, two failed to grow on either of the plastics and one (line 3) grew equally well on both Primaria^TM and NTCP. Of the remaining 9 tumors, five grew selectively or exclusively for NTCP, while four grew exclusively or selectively for Primaria^TM. (Figure 1C).

B. The ovarian tumors

Out of the 10 well differentiated tumors, eight showed growth on Primaria^TM, while four showed growth on NTCP (Figure 2A). Cell lines 1 and 6 did not have growth on either substrate. Four lines grew on Primaria^TM alone and one line showed preferential growth towards Primaria^TM. Two lines had a weak preference for NTCP, and one line produced nearly identical growth between both substrates. Among the 15 poorly-differentiated tumors, lines 8 and 9 showed no growth in either substrate, lines 2, 13 and 14 grew solely in Primaria^TM, and lines 5 and 6 grew solely in NTCP. For those where growth was recorded in both substrates, two lines (1 and 10) were differential for Primaria^TM, three lines (4, 7, and 12) were differential for NTCP, and three lines (3, 7, and 11) showed no preference for either substrate.

IV. Discussion

In this pilot study, we examined the in vitro behavior of endometrial and ovarian tumor cells of different grades to determine if there is a relationship in each tumor type between tumor grades and growth pattern on either Primaria^TM or NTCP. Furthermore, we sought to determine if the growth pattern in endometrial tumors is any different than of those that are ovarian in origin. Our results show that well- and moderately differentiated epithelial tumors of both ovarian and endometrial origin grow better on Primaria^TM than NTCP. We realize that our data is not sufficient to draw any firm conclusions, and the lack of a consistent pattern with regard to cell counts is surprising. The results from low-grade tumor lines however provided a strong indication that Primaria^TM is favored uniformly (Figures 1A, 2A). Without specific genetic analysis to determine metabolic variation in each advanced, high-grade tumor line, we are unable to determine if there is a genetic or metabolic cause for the varied results we recorded in said tumor cells. However, we feel that those tumors which were of medium- or high-grade and selective for growth on Primaria^TM may have future implications with regard to either treatment or prognostic determinations if their final outcomes were tracked and compared to those of the individuals with low-grade tumor pathologies.

It should be noted here that earlier, one of us demonstrated that in variant small cell lung cancer (v-SCLC) the culture in the two plastics separated the tumor types where the cells were different not only with respect to their genomes but also in biology, when they were tested with a panel of eight antibodies (Faruqi and Krueger, 2004). v-SCLC tumor of the lung evolves from small cell to non-small cell lung cancer. Its culture in two plastics separated the tumor into two distinct groups, one growing in one plastic demonstrated highly positive reaction to monoclonal antibodies specific to small lung cancer and the other cell population growing in the other plastic showed a clear negative reaction to the monoclonals (Faruqi and Krueger 2004).

This being a preliminary study our results may be hiding other trends or tendencies that we can not yet ascertain. Because all of the tumor cells used in this study were in vitro in origin, we would have to rely on enzymatic assays to determine whether or not tumor cell metabolism would have been a complicating factor.

Although histopathology is the gold standard in tumor evaluation and prognosis, in certain cases it fails to identify the true genetic potential of a tumor. For example, the portion of a dermoid cyst which was regarded as benign and mature in histopathological evaluation, possessed scores of structurally and numerically altered chromosomes common to the portion which degenerated into malignant immature tumor (Faruqi et al, 1999; Noumoff et al, 2001). By the same token, an immature ovarian teratoma which matured after chemotherapy possessed the same genomic anomalies which were present initially in the immature tumor (Gibas et al, 1993). This culture method has the possibility of adding another dimension to our methods of tumor evaluation.

Figure 1. Endometrial tumor grades showing differential cell growth in normal tissue culture plastic and Primaria^TM. Dividing cell counts are shown along the y-axis of each graph. (A) Low grade endometrial carcinoma cases showing almost exclusive growth in Primaria^TM plastic and very feeble growth in NTCP in one of the three tumors. (B) Moderately differentiated endometrial carcinoma showing more cases with better growth on Pimaria^TM than NTCP. (C) Twelve high grade endometrial carcinoma cases showing two cases without growth on either medium, one case with equivalent growth, four cases preferential (exclusively or selectively) for Primaria^TM and five cases preferential to NTCP.

Figure 2. Ovarian tumor grades showing differential cell growth in normal tissue culture plastic and Primaria^TM. Dividing cell counts are shown along the y-axis of each graph. (A) Low grade ovarian tumors with one showing better while four growing exclusively in Primaria and two grew in neither of the two plastics. (B) Fifteen cases of high grade ovarian tumors grown in Primaria^TM and NTCP show no growth preference for either of the two plastics.

Special note should be made regarding the modified method of culture utilized in this study. Culturing, harvesting and examining the cells on the same plates minimized sampling error by eliminating the process of sampling for cell count.

Thus, every dividing cell was visualized and counted. Use of replicate dishes also reduced the inter-plate variation. Total count of dividing cells should be better than taking samples and counting them manually as it is done in almost all the laboratories with the exception of the currently used automated cell counters (Ionnides, 2003).

Since the only variable evaluated was the grade of the tumor, future studies would obviously require analysis by a variety of factors. The importance of this data is that of the question raised regarding the variability in cell growth; if indeed it is related to selection of specific tumor clones as demonstrated also earlier in v-SCLC (Faruqi and Krueger, 2004).

References

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