American journal of Pathology, Vol. 144, No, 4, April 1994
`Copyright © American Societyfor Investigative Pathology
`
`Variant Sublines of Early-Stage Human
`Melanomas Selected for Tumorigenicity in Nude
`Mice Express a Multicytokine-Resistant
`Phenotype
`
`Hiroaki Kobayashi, Shan Man,
`John R. MacDougall, Charles H. Graham,
`Chao Lu, and Robert S. Kerbel
`From the Division of Cancer Research, Sunnyhroole Health
`Science Centre and Departments ofMedical Biophysics and
`Molecular 6 Medical Genetics University of Toronto,
`Ontario, Canada
`
`Surgical removal of early-stage radial growth
`phase or vertical growth phase primary cutane-
`ous human melanomas usually results in care of
`the disease. Hence there are few examples of
`genetically-related paired human melanoma cell
`linesfor study in which one member ofthepair is
`from a curable early-stage lesion and the partner
`is a more aggressive malignant variant. A rapid
`method ofobtaining such variants is described It
`consists of injecting cellsfrom established early-
`stage radial growth phase or vertical growth
`phase melanoma cell lines—which are normally
`non- or poorly tumorigenic in nude mice—into
`such hosts, where the cell inoculum is co-mixed
`with Matrigel, a reconstituted basement mem-
`brane extract. This resulted in the rapidforma-
`tion ofprogressively growing solid tumors from
`which permanent cell lines were established
`Subsequently,
`the sublines were found to be
`frankly tumorigenic upon retransplantation
`into new nude mouse hosts in the absence ofMa-
`trigel co-injection. This process was repeated a
`second time, resulting in the isolation of second-
`ary sublines, manifesting a stepwise increase in
`tumorigenic properties. The tumorigenic variant
`sublines were examined for their relative sensi-
`tivity to apanel ofdifferent cytokines that are nor-
`mally growth inhibitoryfor melanoma cellsfrom
`early-stage primary lesions. All the sublines were
`found to express an increased resistance to
`the cytokines transforming growth factor-B,
`
`776
`
`interleukin-6, interleukin-1 and tumor necrosis
`factor—a, and did so in a stable manner. Thus the
`results support the hypothesis that a progressive
`multicytokine resistance accompanies the pro-
`gression of human melanoma. The availability of
`such related sublines should provide a valuable
`resource to help study the changes associated
`with, andperhaps causative of; disease progres-
`sion in human malignant melanomas.
`(Am J
`Pathol 1994, 144:776—786)
`
`There are a number of ways in which growth factors
`can contribute directly to the proliferative advantage
`of cancer cells and tumor progression. These include
`the production of stimulatory autocrine growth fac-
`tors,
`increased sensitivity to mitogenic paracrine
`growth factors, and acquisition of resistance to ex-
`ogenous (paracrine) or endogenous inhibitory growth
`factors.1 With respect to the last possibility, the best
`known paradigm is the tendency of cells from most
`types of carcinoma or leukemia to express resistance
`in vitro to the growth inhibitory effects of members of
`the transforming growth factor—B (TGF-B) family, such
`as TGF—B1.2-3 In our laboratory, we have been study-
`ing loss of response to negative growth factors in hu-
`man melanoma. Two
`important
`findings have
`emerged from these studies. First, the number of
`growth factors or cytokines that may function as po-
`tential growth inhibitors of melanocytes or melanomas
`
`
`Supported by grants from the National Cancer Institute of Canada,
`the National Institutes of Health (CA-41233) and the Share Founda-
`tion. HK, JRM, and CL are recipients of a fellowship or Studentship
`from the Medical Research Council of Canada, CHG is a Fellow of
`the National Cancer Institute of Canada and RSK is a Terry Fox Ca-
`reer Scientist of the National Cancer Institute of Canada,
`
`Accepted for publication November 30, 1993.
`Address reprint requests to Dr, Robert S. Kerbel, Division of
`Cancer Research, Reichmann Research Building, 8-218, Sunny-
`brook Health Science Centre, 2075 Bayview Avenue, Toronto. On«
`tario M4N 3M5, Canada.
`GenenteCh 2086
`
`Celltrion v. Genentech
`
`lPR2017-01122
`
`Genentech 2086
`Celltrion v. Genentech
`IPR2017-01122
`
`

`

`777
`Multicytokine Resistance in Variants of Early-Stage Melanoma
`AJPApn'l 1994. Vol. 144. No. 4
`
`may be quite large and include not only TGF-B,4 as
`expected, but also interleukin—1a (IL-1a), lL—6, tumor
`necrosis factor-a (TNF-a) and oncostatin M5 Second,
`resistance to such factors is frequently manifested in
`a seemingly progressive, stepwise manner during
`disease progression.“’7
`Our studies were made possible by virtue of the
`fact that most human melanomas become more ma—
`
`lignant by progression through a series of clinically
`and pathologically well—defined stages of disease?9
`Moreover, cell lines can be established from all these
`
`stages; such lines can then be compared to normal
`melanocytes or atypical nevus derived melanocytes.
`The first stage of tumorigenic melanoma growth is
`the so-called radial growth phase, (RGP) wherein
`primary melanomas grow mainly in the epidermis in
`a plaquelike (horizontal) manner.10 These lesions are
`always curable and seem to consist of tumor cells
`incompetent for metastasis. The next phase is the
`so-called vertical growth phase (VGP); such primary
`tumors begin to grow downward into the underlying
`mesenchymally-derived dermis?9 VGP lesions less
`than 0.76 mm in thickness (thin VGP) are usually
`curable (about 90 to 95% of the time) by surgery, and
`like RGP lesions, are believed to be comprised
`mainly of metastaticaily incompetent melanoma
`cells.8'9 However,
`the thicker VGP lesions ("thick”
`VGP) carry a much worse prognosis and generally
`behave genotypically and phenotypically similar to
`the lesion of distant metastasis?"9 Therefore, RGP
`and thin VGP primary tumors may be considered
`generally as early-stage melanoma, whereas thick
`VGP primary tumors and distant metastases can be
`considered as advanced—stage melanoma.
`The availability of a large number of cell lines es-
`tablished from different stages of melanoma progres-
`sion has proven to be an invaluable tool
`to study
`many aspects of tumor biology as a function of hu-
`man tumor progression?“12 This model neverthe-
`less suffers from one inherent limitation: once an au-
`
`thentic early-stage, primary RGP or early VGP lesion
`has been removed, there is, by definition, rarely a
`follow-up recurrent metastatic tumor that can be re-
`moved from the same patient and studied in a com-
`parative manner with the initial, genetically related,
`lesion. To circumvent this problem, one can use a
`large number of independent cell lines from the dif—
`ferent disease stages, but obtained from different
`patients,
`to study meaningfully the sequential bio—
`logical changes that occur in melanoma cells during
`tumor progression. However, the true relative malig—
`nant status of such cell lines at the time of study is
`frequently impossible to evaluate.
`
`The purpose of the present study was twofold: 1)
`to develop a method of isolating genetically related
`variants of early-stage RGP or VGP primary mela—
`nomas that behave in a much more aggressive fash-
`ion ln vivo, and 2) to use these related cell lines to
`evaluate further the hypothesis that progression of
`melanoma is associated with increasing, stepwise,
`resistance to multiple independent cytokines—a
`phenomenon we have called multicytokine resis-
`tance.7 The strategy we adopted was to inject early—
`stage melanoma cells into nude mice using the Ma-
`trigel-assistance method.13 This is based on the fact
`that certain types of human cancers do not form
`tumors readily, or at all, when injected into nude
`mice, but will do so when the cells are co-injected
`with Matrigel, a reconstituted basement membrane
`extracellular matrix extract.14 This includes, for ex-
`ample, prostatic carcinoma, breast carcinoma, reti-
`noblastoma, and small cell
`lung cancerl3-‘5’19 In
`this regard, early—stage RGP or VGP human melano—
`mas are frequently non- or poorly tumorigenic in
`nude mice‘O-11 (and Dooley T, personal communica—
`tion), but we reasoned that injection of such cells with
`Matrigel might give rise to progressively growing
`solid tumors. Cells from these could then be estab-
`
`lished in culture and subsequently evaluated for tu—
`morigenic capacity (in the absence of Matrigel) and
`for their sensitivity to a number of inhibitory cyto~
`kines.
`
`Materials and Methods
`
`Cell Culture and Establishment of in
`
`Vivo-Passaged Lines
`
`All human melanoma cell lines used in this study were
`maintained in RPMI 1640 (Gibco Laboratories, Grand
`Island, NY) supplemented with 5% fetal bovine serum
`(Hyclone Laboratories, Logan, UT). Three human
`melanoma cell lines (WM35, WM1341 B, WM793) es-
`tablished from early—stage primary lesions by Herlyn
`et al,10 were used in these studies. The clinical and
`pathological characteristics of each line are de—
`scribed elsewhere.“11 Under the conditions we
`
`have employed, two of these cell lines (WM35 and
`WM1341 B) are insufficiently tumorigenic to be able to
`give rise to progressively growing lethal tumors when
`injected subcutaneously (3.0.)
`into athymic nude
`mice (see below for details). ’
`In vivo selection of tumorigenic sublines were es—
`tablished from these three cell lines as follows (see
`Figure 1 for summary using WM35 as an example): in
`the case of the WM35 cell line, 2 X 106 cells were
`
`

`

`Kobayashi et al
`778
`AjP April 1994, Vol. 144, N0. 4
`
`WM 35
`
`(2x106 cells)
`
`TT
`
`82 days
`
`«@415
`
`£15
`
`’D
`
`i...
`
`35-Pl -NI
`
`35- Pl -N2
`
`35- Pl- N3
`
`(2x10 cells)
`
`35-P2-N3
`35-P2-N2
`35-P2-Nl
`Figure 1. Flow diagram summarizing the establishment of in vivo»
`passaged sublines derived from WM35, an early—stage human mela-
`noma cell line. MG: Matrigel. Solid circles represent growing tumors
`in nude mice or tumor cells in culture dishes An identical procedure
`was used to generate variantsfrom the WM134IB and WM793 early-
`stage melanoma cell lines. The tumor volumes (mean : SD) and pe-
`riod of time after inoculation, at which tumors were excised to estab-
`lish each in vivo-passaged sublines, are: 35—P1 sublines, 3057 t
`1139 mmj and 82 days; 35—P2 sublines 2075 t 646. 7 mm3 and 70
`days; 134I—P1 sublines, 458.6 : 148.7 mm3 and 95 days,- 1341492
`sublines, I 725 t 569. 7 mm3 and 70 days; 793-P1 sublines, 1208 1'
`280.6 mm} and 72 days; 793-P2 sublines, 3125 : 221.8 mm5 and
`46 days.
`
`suspended in 0.1 ml of cold serum-free RPMI 1640
`and mixed with an equal volume of cold Matrigel (10
`mg/ml) (Collaborative Research, Bethesda, MD). The
`resulting suspension (0.2 ml of cell suspension con-
`taining 5.0 mg/ml Matrigel) was immediately injected
`so. into the right flank of 5- to 8-week-old female athy-
`
`mic (Swiss) nude mice (Harlen Sprague Dawley). This
`resulted in progressive tumor growth, as summarized
`in the Results section. Eighty-two days after inocula—
`tion, first-passage cell lines (designated 35-P1-N1,
`35-P1-N2, and 35—P1-N3 where “P1" stands for first
`passage and “N" the individual mouse number) were
`adapted to culture by mechanical and enzymatic dis-
`aggregation from three distinct tumors (3057 t 1139
`mm3 mean : SD tumor volume) growing progres-
`sively in independent animals. A total of 2 x 106 cells
`prepared by equal mixing of three first-passage lines
`were co-injected with Matrigel
`into animals in the
`same manner as discussed above. Second-passage
`(P2) lines (designated, for example, as 35—P2—N1 , 35-
`P2-N2, and 35-P2—N3) were established from three
`distinct tumors (2705 : 646.7 mm3 mean : SD tumor
`volume) 70 days after the inoculation.
`Similarly, as described in Figure 1, we established
`the first-passage lines of WM134lB (designated
`1341-P1-N1,
`1341-P1-N2,
`and
`1341-P1-N3) or
`WM793 and the second-passage lines of WM1341 B
`(that is, 1341-P2-N1, 1341-P2-N2, and 1341—P2-N3)
`or WM793. The mean tumor volumes and period of
`time after inoculation, at which tumors were excised
`to establish each in vivopassaged line, are summa-
`rized in the legend of Figure 1. All of these passaged
`variant P1 and P2 sublines were considered as es-
`
`tablished cultured cell lines after being passaged in
`monolayer culture at least 10 times during a period of
`6 weeks and used for experiments.
`
`In Vivo Tumor Growth Assay
`
`To examine the effect of Matrigel on tumorigenicity of
`early-stage human melanoma cells in athymic nude
`mice, various numbers of cultured melanoma cells
`were inoculated so. with Matrigel, as described
`above. Control mice were given a so. injection of the
`same number of cells in 0.2 ml of serum-free RPMI
`
`in the absence of Matrigel. Moreover,
`1640 media,
`various numbers of cells prepared by equal mixing of
`first-passage sublines (Pi-N1, P1-N2, and P1-N3)
`were also inoculated without Matrigel to evaluate the
`effect of in vivo passages on the tumorigenic prop-
`erties of all lines. Mice were monitored up to 3 months
`after inoculation and, if progressively growing tumors
`were observed, the diameters of tumors were meas-
`ured with calipers to calculate the tumor volume from
`the (length X width2/2) as described by Geran et al.20
`At the conclusion of the measurement period, mice
`were sacrificed and their tumors were excised for his-
`
`tological analysis.
`
`

`

`779
`Multicytokine Resistance in Variants of Early-Stage Melanoma
`AJP April 1994, Vol. 144, N0. 4
`
`Southern Blot Fingerprinting Analysis
`
`Growth Assay to Assess inhibitory
`
`We wished to confirm the human genetic origin of the
`in vivopassaged variants, and the absence of sig-
`nificant contamination by mouse—derived DNA ac-
`quired during the tumor growth in nude mice. To do
`this, genomic DNA of each passaged subline
`adapted to culture from its respective growing tumor
`after inoculation with Matrigel was compared with that
`of the parental cultured cell lines by Southern blot
`analysis using mouse or human specific hybridization
`DNA probes Genomic DNA was extracted from the
`parental and in vivo-passaged lines by lysis in 1%
`sodium dodecyl sulphate with subsequent protein
`and RNA digestions. As a control, mouse DNA was
`extracted from spleen tissue obtained from athymic
`nude mice. The resulting DNA (5 ug) was digested
`with EcoRl and size—fractionated in 0.8% agarose
`gels. The separated fragments were immobilized
`onto Zeta-probe blotting membrane (Bio—Rad Labo—
`ratories, Richmond, CA) and membrane probing was
`carried out essentially as outlined elsewhere.21 After
`hybridization with mouse DNA probe, the membrane
`was exposed to x—ray film (Kodak). Hybridization
`using human DNA probe was performed with the
`same membrane after stripping off the mouse DNA
`probe.
`Probes were prepared using the random priming
`method (Pharmacia, Uppsala, Sweden). The mouse
`probe consisted of random primed radiolabeled
`Balb/c genomic DNA digested with BamHI, EcoRl
`and Hindlll restriction enzymes. The human probe,
`cut from plasmid P17H8, consisted of random primed
`radiolabeled DNA specific to the a—satellite sequence
`on chromosome 17, as has been previously de—
`scribed.22
`
`Cytokine Sensitivity
`
`The effects of various cytokines on the growth of each
`cell line were examined by [3H]thymidine incorpora-
`tion assay as described previously?” Five thousand
`cells were seeded in each well of 96—well tissue cul-
`
`ture plates (Nunc, Roskilde, Denmark) with 100 pl of
`ExCell 300 medium (JRH Biosciences, Lenexa, KS)
`supplemented with 1% fetal bovine serum and al—
`lowed to attach overnight. Human recombinant cy-
`tokines were prepared at various concentrations in
`ExCell 300 medium supplemented with 1% fetal bo—
`vine serum and 100 pl of this solution was added to
`each well. Forty-eight hours later, cells were pulsed
`with cell containing 2 uCi of [3H]thymidine in 50 ul of
`serum-free ExCell medium for 4 to 6 hours and then
`
`trypsinized. The DNA was harvested onto filter mats
`(LKB Wallac, Turku, Finland) and [3H]thymidine in-
`corporation was measured with a Betaplate liquid
`scintillation counter (LKB Wallac). Cytokines (pur—
`chased from UBI, Lake Placid, NY) and their concen-
`trations used were lL-6 (0.5—25 ng/ml), TGF-m
`(0.25—5 ng/ml),
`IL-1a (0.5—5 ng/ml), and TNF-a
`(0.5—25 ng/ml). [3H]thymidine incorporation in con-
`trols without added factors was considered as 100%.
`
`Results
`
`Effect of Co-injection with Matrigel on
`
`Tumor Growth of Early-Stage Melanoma
`Cell Lines
`
`Aftertumor cell inoculation, all animals were observed
`for 3 months, or until their tumors reached a maximum
`of approximately 2 cm in diameter. Table 1 shows the
`
`Table 1. Effects ofMatrigel and in viva Passage on Tumon’gem‘city in Nude Mice of Early—Stage Human
`Melanoma Cell Lines
`
`Tumor-take incidence‘
`
`Number of
`inoculated
`WM35
`35—P1 fl 1341—P1 & 793-191
`cells
`—MG
`+MG
`—MG
`—MG
`+MG
`—MG
`—MG
`+MG
`—MG
`
`
`5/5
`5/5
`3/5
`5/5
`5/5
`0/5
`5/5
`5/5
`1/10
`2 x 106
`5/5
`5/5
`2/5
`5/5
`5/5
`0/5
`5/5
`5/5
`0/5
`1 x 106
`ND
`ND
`ND*
`5/5
`5/5
`0/5
`5/5
`5/5
`0/5
`5 x 105
`4/5
`5/5
`1/5
`4/5
`5/5
`0/5
`3/5
`5/5
`0/5
`1 X 105
`2/5
`4/5
`0/5
`4/5
`5/5
`ND
`3/5
`4/5
`ND
`1 x 104
`
`1 X 103 1/5 ND 2/5 1/5 ND 5/5 1/5 0/5 4/5
`
`
`
`
`
`
`
`
`
`' Tumor development and growth was observed until 3 months after so. inoculation of various numbers of parental or in vivopassaged
`(P1) cell
`lines into nude mice in the presence (+MG) or absence (—MG) of Matrigel. The cell lines designated as 35»P1, 1341-P1, and
`793-P1 refer to the first-passage sublines derived from primary tumors after inoculation of the parental WM35, WM1341. or WM793, respec-
`tively, with Matrigel, as described in Materials and Methods.
`T ND, not determined.
`
`

`

`Kobayashi et al
`780
`AjP April 1994, Vol. 144, No. 4
`
`tumorigenic profiles of several parental early—stage
`melanoma lines or in vivopassaged sublines (first
`passage), injected with or without Matrigel into nude
`mice.
`It is obvious by comparing the tumor-take in-
`cidence of parental (nonpassaged) tumor lines, with
`or without co—injection of Matrigel, that Matrigel sig-
`nificantly enhanced the tumor-forming ability of all the
`cell lines. In particular, of the three cell lines, the tu—
`morigenicity of WM1341 B line showed the highest en-
`hancement by Matrigel: remarkably, a 100% tumor—
`take incidence was obtained by the so. inoculation of
`no more than 103 tumor cells co-injected with Matri—
`gel, even though no tumors developed after 2 x 106
`tumor cells were inoculated without Matrigel. With
`both WM35 and WM793 lines, the tumor-take inci—
`
`dence by the inoculation of 103 cells with Matrigel
`(40% and 80%, respectively) was higher than that by
`the injection of 2 X 106 cells without Matrigel (10%
`and 60%, respectively).
`
`Morphological and Genetic Preservation
`
`of Tumor Characteristics during in Vivo
`
`Passages
`
`By hematoxylin and eosin staining, all tumors derived
`from a parental WM35 line inoculated so. with or with—
`out Matrigel and from first—passage WM35 lines
`
`grown in the absence of Matrigel showed similar mor-
`phological appearances, typical of human melanoma
`(Figure 2, A to C). Similarly, neither WM793 tumors
`arising in Matrigel—injected animals nor tumors de-
`rived from in vivopassage (P1) sublines showed a
`significant change in morphological pattern com-
`pared to the parental WM793-derived tumor in the
`absence of Matrigel (Figure 2, D to F).
`Figure 3 shows a DNA fingerprinting assay by
`Southern blot analysis using mouse (Figure 3A) or
`human (Figure 38) specific DNA probes. The DNA
`sample derived from mouse spleen tissue probed
`with random primed mouse genomic DNA resulted in
`a smear consisting of numerous bands (Figure 3A,
`lane 1). On the other hand, no bands with the mouse
`probe were seen in the lanes loaded with DNA
`samples from the cultures of in vivopassaged (P1)
`sublines as well as the parental early—stage human
`melanoma cell lines (Figure 3A, lanes 3 to 14). When
`probed with a random primed human DNA probe
`specific to the a—satellite sequence on chromosome
`17 (Figure 38), P1 sublines of WM793 (lanes 4 to 6)
`and WM35 (lanes 8 to 10) showed exactly the same
`patterns of bands as their parental WM793 (lane 3)
`and WM35 (lane 7) cell lines, respectively. In vivo-
`passaged WM1341B sublines (lanes 12 to 14) dis—
`played similar but not identical patterns of bands
`when compared to the parental WM1341B cell line
`
`
`
`Figure 2. Histological appearance of tumor tissues derivedfrom parental (nonpassuged) human melanoma cell lines and their in vivofiassaged
`(PI) sublines. inoculated with or without Matrigel. Some tumor specimens were obtained after 5.c. inoculation of 2 X 10" cells ofparental early—
`xlage melanoma cell lines with ( WM35: B,- WM793: E and H,- WM13413: I) or without ( WM35: A; WM793: D) Matrigel. Others were obtained by
`injection on X 10" cells offirst—passage (Pl) sublines in the absence of Matrigel (SS—P1: C and G.- 793—P1; F). A to F: hematoxylin and eosin
`xtaintng. Note that these three distinct tumors derived from WM35 (A to C) or WM793 (D to F) cell lines xhow a similar morphological pattern
`(mica! of human melanoma.
`
`

`

`781
`Multicytokine Resistance in Variants of Early-Stage Melanoma
`AjP April 1994, Vol. 144, N0. 4
`
`A
`
`l23456789l011121314
`
`
`
`
`12345678910lll21314
`
`B
`
`Figure 3. DNA fingerprinting profiles of early-slage human mela—
`noma cell lines and tbeir in vivo-passaged variants. DNA samples ex-
`tractedfrom monolayer cultures ofparental (nonpassaged) cell lines
`and in vivo-passaged sublines were probed with random primed
`mouse genomic DNA (A). The same membrane was used for hybrid»
`ization with random primed human DNA specific to the msarellite se-
`quence on chromosome 1 7 after stripping off the mouse DNA probe
`(B). Lane 1: mouse DNA extracted from spleen tissue obtained from
`athymic nude mice ( control),- lane 2: empty; lane 3: parental WM793
`line,- lanes 4 to 6: first-passage sublines of WM793 ( 793»P1-N1, 793—
`PI—N2, and 793—P1—N3,
`respecliziely); lane 7: parental WM35 line:
`lanes 8 to 10: first-passage sublines of WM35 (35—P1—N1, 35‘PI-N2,
`and 35-PI»N3, respectively),- lane 11: parental WM134IB line: lanes
`12 lo 14;firsI-passage sublines of WM134IB (1341—P17N1, 134I-P1»
`N2, and 1341—P1—N3, respectively).
`
`(lane 11): in the sublines several different sized bands
`were apparent, which suggested some DNA-
`rearrangements had occurred during in vivo pas-
`sages. However, the pattern observed in lane 12
`(1341-P1-N1) indicated that this cell line may be a
`cross—contaminant of WM35 or WM793 cells, or one
`of their selected sublines. Because of its equivocal
`origin, this cell line was not included in further studies.
`Thus, by using mouse or human DNA probes, we con-
`
`firmed that the in vivopassaged sublines were in—
`deed human in origin, and in all likelihood, were de-
`rived from the parental cell lines used to inoculate the
`mice and were not contaminated by host-derived
`mouse DNA.
`It is also apparent that this procedure
`may be useful
`in picking up occasional cross-
`contaminating cell lines.
`
`Evidence of Tumor Progression during in
`Vivo Passages: Increased Tumor Growth
`Properties in Passaged Cell Lines
`
`By comparing the tumor-take incidence between pa-
`rental (nonpassaged) early-stage melanoma lines
`and in vivo-passaged sublines after so. inoculation in
`the absence of Matrigel, it was apparent that in vivo-
`passage resulted in the emergence of sublines hav-
`ing a highly elevated tumor-forming ability and that
`this was independent of the promoting effect pro-
`vided by Matrigel (Table 1). During the 3-month pe-
`riod of observation, at least 2,000-fold and 100-fold
`fewer cells of first—passaged lines were required to
`produce tumors with similar tumor—take incidence by
`parental WM35, WM1341 B, and WM793 cell lines, re-
`spectively (see Table 1).
`Because a 100% tumor—take incidence of each cell
`line was obtained 3 months after so. inoculation of
`
`106 tumor cells with Matrigel, we compared tumor
`growth curves among parental and in vivo-passaged
`cell lines. As shown in Figure 4, all of in vivopassaged
`variants acquired highly enhanced tumor-forming
`ability Greater tumor growth was seen as the in vivo-
`passage number increased, especially in the case of
`a series of WM1 341 B cell lines: the tumor volume dou-
`
`tumors derived from the parental
`bling time of
`WM1341B cell line was 29.3 days; for 1341—P1 sub-
`lines, 18.7 days; for 1341-P2 sublines, 12.8 days; for
`the parental WM35 cell line, 17.5 days; for 35-P1 sub—
`lines, 13.6 days; for 35—P2 sublines, 11.9 days; for the
`parental WM793 cell line, 17.5 days; for 793—P1 sub-
`lines, 11.2 days; and for 793—P2 sublines, 10.0 days.
`
`Multicytokine Resistance in Human
`Melanoma Cells Is Acquired through in
`
`Vivo Passages
`
`the growth of
`We have previously reported that
`WM35 early-stage melanoma cell line is inhibited in
`vitro by IL-6,
`lL—1a, TNF-a, TGF—B1, and oncostatin
`M, whereas the majority of advanced stage human
`melanoma cell lines we examined were found to be
`
`partially or completely resistant to these inhibitory
`effects.“‘6 Therefore,
`the sensitivity to these cyto-
`
`

`

`Kobayashi et al
`782
`AjP April 1994, Vol. 144, No. 4
`
`10000
`
`WM35
`
`WM1341B
`
`WM793
`
`
`
`
`
`TumorVolume(mm3)
`
`1000
`
`100
`
`17.54 days
`
`
`
`
`11.87 days
`
`29.28 days
`
`\
`
`i/l
`
`18.73 days
`
`12.84 days
`
`17.48daysx/I/E
`
`\
`
`11.24 days
`
`10.00 days
`
`
`
`
`
`Weeks after Inoculation
`Figure 4. Effects of in vivo passage on the tumor growth properties of early-stage human melanoma lines in athymic nude mice. 0.1 ml cell sus-
`pension containing 1 million cells was injected with the same volume of Matrigel (10 mg/ml) into five nude mice per group. All mice developed
`tumors and results represent the mean tumor volume I SEM calculated as described in Material and Methods. 0: parental line: C, first—passage
`line(PI); A; secondepassage line (P2). A.- WM35 cell lines; B: WM134IB cell lines; C: WM793 cell lines.
`
`kines (with the exception of oncostatin M) was com—
`pared between parental WM35 cells and in vivo-
`passaged sublines (P1 and P2 lines) by using the
`[3H]thymidine incorporation assay. Shown in Figure
`5 is a representative result for each cytokine from
`several tests using various concentrations of lL—6
`(0.5—2.5 ng/ml), TGF-Bf (0.5—2.5 ng/ml), lL—1a (0.5—
`2.5 ng/ml), and TNF—a (0.5—5 ng/ml). All of the P1 and
`P2 sublines showed a statistically significant
`in-
`crease of [3H]thymidine incorporation after 48 hours
`exposure to 2.5 ng/ml of lL-6 (Figure 5A), TGF—Bf
`(Figure SB), and lL-1a (Figure 5C) or 1 ng/ml of TNF-a
`(Figure 5D) as compared with that of parental WM35
`cell lines. Data from experiments using other concen-
`trations of
`the four cytokines showed the dose—
`response inhibitory effects of cytokines to each line,
`but the differential cytokine sensitivity between pa-
`rental and in viva-passaged cell lines was consistent
`with the data shown here.
`The WM1341 B cell line is not sensitive to lL-1a and
`
`TNF-a, but is inhibited by exposure to lL-65 and TGF-
`[31.4 Hence, the change in sensitivity to cytokines dur—
`ing in vivo passages was examined using lL-6 and
`TGF-Bf (Figure 6). Similar to the WM35 cell line and
`its sublines, every in viva-passaged WM13418 sub-
`
`line (P1 and P2 lines) manifested a significantly lower
`sensitivity to 5 ng/ml lL-6, compared to the sensitivity
`of the parental WM1341 B line (Figure 6A). Most of the
`passaged WM1341B sublines, except 1341-P2-N1,
`were statistically more resistant to 5 ng/ml TGF-Bt
`than the original WM1341B line (Figure BB). At other
`concentrations of lL-6 (1—25 ng/ml) or TGF-m (0.5—5
`ng/ml), similar resistance to each cytokine was seen
`in the in vivopassaged WM1341 B sublines (data not
`shown).
`Because lL-6 has no inhibitory effects on the
`growth of the WM793 Iine,5 TGF-Bf, lL—1a, and TNF—a
`were used to check for the acquired cytokine resis-
`tance by the in viva-passaged WM793 sublines (P1
`and P2 lines) (Figure 6). After 48 hours exposure to
`0.5 ng/ml TGF—Bf,
`four of six in vivopassaged
`WM793 sublines showed a significant increase in
`[3H]thymidine incorporation rates, as compared with
`that of the parental WM793 line (Figure 6C). Statisti-
`cally significant resistance to 1.25 ng/ml IL-1a was
`seen in four of six in vivopassaged WM793 sublines
`(Figure 6D). At other concentrations of TGF-[i (0.25 to
`2.5 ng/ml) or lL-1a (0.5 to 5 ng/ml), we obtained com-
`parable results (data not shown). In the case of ex-
`posure to 2.5 to 25 ng/ml of TNF-a, the differential
`
`

`

`783
`Multicytokine Resistance in Variants of Early-Stage Melanoma
`AjP April 1994, Vol, 144, N0. 4
`
`IL 1a 2.5ng/ml
`
`TNF-a 1nglml
`
`120
`
`100
`
`80
`
`50
`
`40
`
`20
`
`°
`
`120
`
`100
`
`80
`
`so
`
`4°
`
`2°
`
`0
`
`g E g 2 E g 2
`2 ,'_ _'
`"_
`<<l
`<<l
`({l
`g D.
`0|- D. D.
`[L
`a.
`“5)
`ID
`Lt.)
`“'3
`It.)
`It!)
`mmmmmm
`
`(“2.J 2 E 2
`8 E (2
`n
`I
`“I,
`({l
`E ‘I_
`I
`g [L E E a D.
`[L
`“'3
`“I,
`II.)
`If)
`I},
`III)
`commmmm
`
`sensitivity was not as obvious between in vivo—
`passaged sublines and parental WM793 line (data
`not shown).
`
`Discussion
`
`The results of our studies contain two potentially im-
`portant findings. First,
`it is possible to obtain tumors
`rapidly in nude mice from injections of early-stage
`human melanoma cell lines, provided the cells are
`co-injected with Matrigel; moreover, cell lines estab-
`lished from these tumors readily engratt and grow
`progressively when injected into new nude mouse re-
`cipients in the absence of Matrigel. Second, virtually
`all the tumorigenic variants obtained from different
`early—stage melanomas we examined manifested in-
`creased resistance to the growth-inhibitory effect of a
`panel of cytokines, including TGF—Bl, TNF—a, IL-1a,
`and lL-6. Thus, the results support the hypothesis that
`acquisition of multicytokine resistance7 accompanies
`human melanoma progression. Expression of such a
`phenotype may be an effective way for tumors to ac-
`
`quire a growth advantage and give rise to tumor sub-
`populations of increasing aggressiveness.7
`With respect to our Matrigel results, injection of hu-
`man tumor cells with this extract seems to be an ef-
`
`fective procedure to obtain tumor growth in nude mice
`using various types of tumor that are normally very
`poorly tumorigenic, or completely unable to grow in
`such hosts. This was first discovered by Fridman et al
`using human small cell carcinoma cell lines.“15 It
`has since been confirmed by others using human tu-
`mors
`such as prostate cancer,”18 retinoblas—
`tomaJG'19 and breast cancer.19 Whereas malignant
`melanomas are normally among the most tumorigenic
`of all human cancers in nude mice, this does not apply
`to cell lines from the early-stages of this disease.10
`Thus, Herlyn and his colleagues have reported that
`cell lines established from RGP or early VGP primary
`tumors (removed from the patients who did not ex—
`perience recurrent disease, ie, were cured by surgi—
`cal excision of the tumors), do not generally grow
`readily when injected into nude mice.10 A similar ob—
`servation has been noted by Dooley using six newly
`
`120
`
`lL-6 2.5nglml
`
`C 100
`
`80
`
`60
`
`O 4
`
`:
`
`COQ
`
`I..—
`
`TGF-B 2.5n9/ml
`
`40
`
`<3
`o\
`V 20
`G)
`
`x c
`
`s
`4—:
`Q.
`
`0
`
`:3 120
`(D
`C 100
`Figure 5. [‘I-Ilthymidine incorporation (growth)
`'—
`assay for cytohine sensitivity. Cells were plated
`__
`at 5 X 10‘ cells/well in 96-well plates, allowed
`E 80
`to attach overnight and incubated with or
`without various concentrations of cytoleines
`(0,5 to 2.5 ng/ml oflL-6, TGF-BI, and IL-Ia or >
`045 to 5 ng/ml of mpg). After 48 hours, [‘HI-
`.C 60
`thymidirie incorporation into DNA was meas-
`'—
`ured. Representative data are shown at concen-
`l
`trations of 2,5 rig/ml of lL-6 (A), TGF—BI (B). A 4°
`and IL-Iix (C) or 1 ng/ml of 7NF-a (D). Data I
`are expressed as mean 1 SEM of triplicate de-
`(0"
`terminations compared with the controls un-
`treated of each cell line (which was considered
`as 100%), WM35: parental earlyestage human
`melanoma cell line; 35-P1»N1,
`-N2 and —N3.-
`first-passage sablines: 35»P2—N1, NZ. and -N_3.-
`second-passage sablines. Probability was calcu-
`lated brStudent’st-tesl. Parental W35 line as
`each passaged subline in A, B, C and D, P <
`001.
`
`20
`
`0
`
`

`

`TGF-fl1 0.5ng/ml
`
`_
`IL 101 1.25ng/ml
`
`120
`
`100
`
`so
`
`60
`
`40
`
`20
`
`0
`
`120
`
`100
`
`80
`
`60
`
`40
`
`20
`
`0
`
`8 E g ('2) E g 2
`" 1'—
`.'— 1'- o':
`({i
`d:
`2 a.
`n_
`n_ o. n.
`n_
`g 0!,
`(5
`0:,
`(4-,
`c5,
`(,3
`C)
`CD O) O) O) O)
`" " " " i‘ N
`
`l‘ll/Ihvmidine incorporation (may
`Figure 6.
`fur cv/oleine senxitit'iiv, Cells were plated at S X
`10‘ cells/well in 96-well plates. allowed to at-
`lach overnight and incubated with or without
`various concentrations of cvloleines (1 to 25
`ng/ml IL-6 and 0.5 to 5 ng/ml TGF—fil for the
`Wit/113418 cell lines: 0.25 to 2.5 Hg/flll TGF-BI,
`and 0.5 to 5 ng/ml IL-Ia for the “711/1795 Cell
`lines). After 48 hours, l‘H/lhymidine incorpora-
`tion into DNA was measured. Representative
`data are shown at concentrations of 5 ng/ml of
`lL-6 (A) and TOW-BI (B) for Wit/[13418 cell
`lines and 05 ng/ml TGF—fil (C) and 1.25
`ng/ml lL-Ia (Di/or WM795 cell lines. Data are
`expressed as mean : SEM of triplicate determi-
`nations compared with the controls of each cell
`line (considered at 100%), WM134IB and
`WM795: parental early-stage hmnan mela-
`noma cell lines: 134I-PI»N1,-N2.-N5 and 793-
`PleNI.-N2,7N3.~ firstapassage sub/mes,- 1541472